Methods of inducing regulated pancreatic hormone production in non-pancreatic islet tissues

ABSTRACT

Disclosed are methods and pharmaceutical compositions for inducing pancreatic hormone production.

RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 09/584,216,filed May 31, 2000, which claims the benefit of U.S. Ser. No. 60/137,143filed Jun. 1, 1999 and U.S. Ser. No. 60/198,532 filed Apr. 19, 2000 andclaims the benefit of U.S. Ser. No. 60/469,715 filed May 12, 2003. Thecontents of these applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

This invention relates generally to methods of inducing a pancreaticendocrine phenotype and function including pancreatic hormone productionin a non-endocrine tissue and in particular to methods andpharmaceutical compositions for treating endocrine related disorders.

BACKGROUND OF THE INVENTION

The endocrine pancreas consists primarily of islet cells that synthesizeand secrete the peptide hormone glucagon, insulin, somatostatin andpancreatic polypeptide. Insulin gene expression is restricted topancreatic islet β-cells of the mammalian pancreas through controlmechanisms mediated in part by specific transcription factors. In othercells the insulin, other pancreatic hormones and specific peptidasesgenes are trancriptionally silent. The homeodomain protein PDX-1(Pancreatic and Duodenal Homeobox gene-1, also known as IDX-1, IPF-1,STF-1 or IUF-1) plays a central role in regulating pancreatic isletdevelopment and function. PDX-1 is either directly or indirectlyinvolved in islet-cell-specific expression of various genes such as forexample insulin, glucagon somatostatin, proinsulin convertase 1/3(PC1/3), GLUT-2 and glucokinase. Additionally, PDX-1 mediates insulingene transcription in response to glucose.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that ectopic expressionof pancreatic and duodenal homobox gene 1 (PDX-1) in liver induces theexpression of the silent pancreatic hormone genes and the processingmachinery, which converts the prohormones into mature biologicallyactive hormones.

The invention provides methods of inducing the expression of apancreatic gene in a cell by introducing to the cell a PDX-1 inducercompound. The invention further provides a method of converting anon-pancreatic cell to a pancreatic cell, by contacting thenon-pancreatic cell with a PDX-1 inducer compound. The non-pancreaticcell is contact with the PDX-1 inducer compound in an amount to inducethe expression of endogenous PDX-1, an embryonic marker, insulin,glucogon, or somatostatin in repress the expression of C/EBPβ, albuminor ADH-1 in said non-pancreatic cell. An embryonic marker is forexample, alpha-1 fetoprotein or Gata-4.

A PDX-1 inducer compound is any compound that induces the expression ofendogenous PDX-1. A PDX-1 inducer compound is a nucleic acid,polypeptide or small molecule. Exemplary PDX-1 inducer compoundsinclude, a nucleic acid encoding a pancreatic and duodenal homobox 1(PDX-1) polypeptide, a neuroD polypeptide or a betacellulin polypeptide.

The nucleic acid is operably linked to a promoter such as for example,cytomegalovirus (CMV) promoter, a BOS promoter, a transthyretinpromoter, a glucose 6-phosphatase promoter, an albumin intestinal fattyacid binding protein promoter, a thyroglobulin promoter, a surfactant Apromoter, a surfactant c promoter or a phosphoglycerate kinase 1promoter. The method of nucleic acid is present in a plasmid or avector. The vector is a viral vector such as an adenovirus vector or alentivirus vector. The adenovirus vector is for example a gutlessrecombinant adenovirus vector.

By “induces the expression” it is meant that expression of the gene isincreased in the presence of the compound compared to the absence of thecompound. to said cell a composition comprising a nucleic acid encodinga pancreatic and duodenal homobox 1 (PDX-1) polypeptide, in an amountsufficient to induce said gene expression in said cell. By “repressesthe expression” it is meant that expression of the gene is decreased inthe presence of the compound compared to the absence of the compound.

A pancreatic gene includes for example a pancreatic transcription factorsuch as PDX-1, beta 2, ISL-2, NRx6.1, Ngn3.1, or NKx2.2, an endocrinegene such is SCG2, SGNE1, CHGN, PTPRN, AMPH, NBEA, NeuroD or folistatinor an exocrine gene such as serine protease inhibitor, Kazal type 1,Elastase, factor-p48, regenerating islet-derived 1 alpha.

The cell is provide in vivo, in vitro or ex vivo from a mammaliansubject. The cell is a non-pancreatic cell. The cell is a differentiatedcell. The cell is an ectodermal cell, endodermal cell, or mesodermalcell. For example the cell is a liver cell, a skin cell, or a bonemarrow cell. Optionally, the cell is further contacted with atransfection agent or a composition containing nicotinamide, epidermalgrowth factor, activin A, hepatic growth factor, exendin, GLP-1 orbetacellulin.

The invention provides methods of inducing pancreatic hormone, e.g.,insulin, glucagon and somatostatin levels in a subject. In one aspect,the method includes administering to a subject in need thereof acompound which increases PDX expression or activity in an amountsufficient to induce pancreatic hormone production in the subject. Inanother aspect, the method includes providing a cell capable ofexpressing a pancreatic hormone, contacting the cell with a compoundwhich increases PDX expression or activity and introducing the cell intoa subject, thereby inducing pancreatic hormone production in thesubject.

Also provided in the invention is a method of treating, alleviating asymptom of or delaying the onset of a pancreatic-related disorder suchas diabetes, e.g., Type I or Type II in a subject. The method includesadministering to a subject a therapeutically effective amount of acompound which increases PDX expression. For example the compound anucleic acid encoding a pancreatic and duodenal homobox 1 (PDX-1)polypeptide. Symptoms of diabetes include hyperglycemia, elevated bloodglucose (blood sugar), frequent urination excessive thirst, extremehunger, unusual weight loss, increased fatigue, irritability, or, blurryvision. Diabetes is diagnosed for example by fasting plasma glucose testor random blood glucose test.

In another aspect the invention provides a method of inducing apancreatic islet gene expression profile in a subject. The methodincludes administering to a subject in need thereof a compound whichincreases PDX expression or activity in an amount sufficient to inducepancreatic islet gene expression.

In yet a further aspect of the invention is a method inducing orenhancing a pancreatic islet cell phenotype in a cell. The methodincludes contacting a cell with compound which increases PDX expressionor activity in an amount sufficient to induce or enhance pancreaticislet cell phenotype in said cell.

Also included are pharmaceutical composition that includes a compoundwhich increases PDX expression and a pharmaceutically acceptablecarrier.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration demonstrating detection of mRNA in Balb/c miceliver tissue for mouse insulin I (mI-1), mouse insulin II (mI-2), humaninsulin, PDX-1 and β-actin after adenovirus treatment as determined byRT-PCR. Lane 1: no DNA (negative control for PCR); lanes 2-6: liversfrom AdCMV-PDX-1 treated mice lanes 7, 8: livers fromAdCMV-PDX-1+AdRIP-1-hIns-treated mice; lanes 9-11: livers from controlAdCMV-β-gal+AdRIP-1-hIns-treated mice; lanes 12, 13: livers fromAdCMV-hIns-treated mice; lane 14: normal mouse pancreas.

FIG. 2 is an illustration of the HPLC elution profiles of insulinrelated peptides extracted from murine tissue. Panel A shows the profilefrom the pancreas of a PDX-1 treated mouse. Panel B shows the profilefrom the liver of a PDX-1 treated mouse.

FIG. 3 is an illustration demonstrating detection of mRNA for PDX-1,somatostatin Somato), proinsulin convertase PC1/3 (PC1/3), glucagon(Glucg) and β-actin determined by RT-PCR: Total RNA extracted from PDX-1and control treated mice was reverse-transcribed using a PC1/3 specificprimer. lanes 1-3: mice treated by AdCMV-PDX-1; lanes 4-5: mice treatedby AdCMV-β-gal; lane 6: pancreas; lane 7: no cDNA, (control for PCR).

FIG. 4 is an illustration demonstrating ectopic PDX-1 expression in micelivers ameliorates STZ induced hyperglycemia: C57BL/6 males at 12-13weeks were treated by 220 mg/kg STZ in citrate-buffer. 36-48 hour afterSTZ treatment mice were injected by AdCMVPDX-1 (n=15 mice), or ascontrol by AdCMVβ-gal (n=22, however, 12 died 3-5 days after STZtreatment, additional 3 mice died 6-7 days after STZ treatment). Nomortality occurred upon AdCMVPDX-1 treatment. Each treatment includedsystemic injection of 2×10⁹ PFU (plaque forming units) of recombinantadenovirus in 200 μl saline. Glucose levels were determined in bloodsamples drawn from the ocular vein.

FIG. 5 is an illustration demonstrating ectopic PDX expression in maturehepatocytes in culture activates insulin promoter (rat insulin-1promoter), co-delivered to the same cells by AdRIPhIns. Human insulin isdetected as in FIG. 1. Lane 1: cells treated by AdCMV PDX-1+AdRIP hIns,lane 2: AdCMVβ-galactosidase+AdRIP hIns, lane 3: Control.

FIG. 6 is an illustration demonstrating the induction of Insulin 1 andSomatostatin gene expression in primary monolayer cultures of fetalFisher rat (E14) hepatocytes. Fetal hepatocytes were isolated fromFisher 344 rat embryos at day 14 of gestation, and plated on collagencovered tissue culture dishes. Cells were infected by AdCMVPDX-1 at 2-5MOI (multiplicity of infection=number of viral particles per cell).Total RNA was extracted from the culture 4 days after viral treatmentand was analyzed for somatostatin gene expression by RT-PCR. RNA wasreversed transcribed as in FIG. 1 using oligo (dT)₁₅ primers andamplification by PCR was performed using primers and conditions aselaborated in Table 1. Lanes 1-3: samples from cells treated by PDX-1,lanes 4-6: untreated samples (control) lane 7: no DNA, PCR products wereresolved on 1.7% agarose gel electrophoresis.

FIG. 7 is an illustration demonstrating the effect of effect of glucoseon PDX-1 activation, manifested by its increased binding capacity to theinsulin promoter. GLUT-2 and GK expression promote this activation byallowing glucose entry and metabolism. RIN-38 cells of intermediatepassage were infected by AdCMV-GLUT2 or AdCMV-GK (lanes 4-6) or were nottreated (lanes 1-3). 24 hours after viral treatment all cells wereincubated in 0.2, 5 and 15 mM glucose.

FIG. 8A is a photograph of RT-PCR analysis showing ectopic PDX-1expression induces an endocrine repertoire of pancreatic gene expressionin mature liver in-vivo.

FIG. 8B is a photograph of RT-PCR analysis showing ectopic PDX-1expression induces an exocrine repertoire of pancreatic gene expressionin mature liver in-vivo.

FIG. 9 is a photograph of RT-PCR analysis of pancreatic gene expressionin PDX-1 treated livers as a function of time after one singleadministration of Ad-CMV-PDX-1, in-vivo.

FIG. 10 are a series of photographs showing insulin and glucagonpositive cells are located in the proximity of central veins (cv) fourto six months after treatment. Panel A Insulin; Panel B glucagon 120days; Panel C Insulin positive cells 180 days after Ad-CMV-PDX-1administration; Panel D control.

FIG. 11A is a scatter plot showing hepatic insulin content in individualmice as a function of time after systemic Ad-CMV-PDX-1 administration:at 56 days (PDX-1 treated, n=3, control, n=6), 120 days (PDX-1 treated,n=7, control, n=3) and 180 days (PDX-1 treated, n=4, control, n=5).Hepatic IRI content in PDX-1 treated (□) or control (♦) mice arepresented separately for each individual mouse.

FIG. 11B is a bar chart showing hepatic glucagon content (PDX-1 treated,n=10, control, n=10) in Ad-CMV-PDX-1 treated mice.

FIG. 11C is a bar showing hepatic somatostatin (PDX-1 treated, n=9,control, n=7) in Ad-CMV-PDX-1 treated mice.

FIG. 12A is a photograph showing PCR of ectopic rat PDX-1 cDNA attachedto CMV promoter, which reflects of the presence of viral Ad-CMV-PDX-1DNA in treated liver as a function of time (in days) after adenoviraladministration to mice in-vivo.

FIG. 12 B is a photograph showing RT-PCR analysis of rat PDX-1 (rPDX-1),mouse PDX-1 (mPDX-1) and β-actin gene expression in liver, as a functionof time after Ad-CMV-PDX-1 administration, in-vivo.

FIG. 12C is a bar chart showing quantitation of ectopic (rat) vs.endogenous (mouse) PDX-1 expression as a function of time after initialAd-CMV-PDX-1 treatment using real time PCR. Mouse PDX-1 (stripedcolumns) and rat PDX-1 (solid black columns).

FIG. 13 is bar chart showing hepatic insulin production protects micefrom STZ induced hyperglycemia, eight months after the initialAd-CMV-PDX-1 treatment, and that transdifferentiated insulin producingcells in liver are resistant to STZ.

FIG. 14 is a bar chart depicting insulin gene expression in humankeratinocytes, wherein PDX-1 treatment works in a dose dependent manner:100 but not 10 moi (multiplicity of infection) were capable ofactivating insulin gene expression.

FIG. 15. is a bar chart depicting glucagon gene expression in humankeratinocytes.

FIG. 16. is a bar chart depicting somatostatin gene expression in humankeratinocytes.

FIG. 17A is an illustration depicting an Ad-RIP-GFP-CMV-PDX-1 construct,this construct allows identifying cells that ectopic PDX-1 expressioninduces the activation of ectopic insulin promoter.

FIG. 17 B is a series photographs showing phase contrast morphology (A,C), and green fluorescence (B, D) of adult liver cells at passage 2 (A,B) and at passage 8 (C, D) infected by Ad-RIP-GFP-CMV-PDX-1(magnification×200).

FIG. 17C is a bar chart showing the number of adult (black bars) andfetal (grey bars) liver cells that exhibit insulin promoter activationmanifested by green fluorescence as a function of the passage numberin-vitro (n≧20 random fields were counted at each passage).

FIG. 17D is a bar chart showing the transdifferentiation potential(lined bar) of adult liver cells is calculated as the percent of cellsthat exhibit the insulin promoter activation (Ad-RIP-GFP-CMV-PDX-1,black bar) divided by the total cell infection capacity, exhibited byfluorescing cells subsequent to Ad-CMV-GFP infection (grey bar, n>10),as a function of the passage number.

FIG. 18A is a bar chart showing pancreatic hormones gene expression inadult and fetal liver cells treated by Ad-CMV-PDX-1 with or withoutgrowth factors (GF) supplementation. Ad-CMV-hIns serves as positivecontrol for insulin gene expression. C_(t) (threshold cycle) values wereall normalized to β-actin gene expression within the same RNA sample.

FIG. 18 B is a quantitative RT-PCR (Real-time) amplification curve ofInsulin in human islets (RNA diluted 1:50, A), in Ad-CMV-PDX-1 and GFtreated adult (B) and fetal (C) human liver cells, untreated adult (D)and fetal (E) liver cells (all with the same Ct-values for β-actin geneexpression). The curves are presented as the ΔRn (fluorescentic units)versus the cycle number of the amplification reaction. Showing thatmature liver cells are as efficient as fetal human liver cells inactivating insulin gene expression in response to PDX-1

FIG. 19 is a bar chart showing insulin content, secretion and processingin adult primary liver cells that were treated by Ad-CMV-PDX-1,supplemented by growth factors and analyzed for insulin content (blackbar; n≧10), insulin (lined bar; n≧25) and C-peptide (pointed bar; n≧25)secretion by static incubations for 48 hours. Ad-CMV-hIns infected cellsserve as positive control for non regulated and unprocessed insulinsecretion. Results indicate fold of increase (FOI) compared to untreatedcontrol liver cells.

FIG. 20A is a series of photographs showing insulin secretory granulesin PDX-1 treated adult human liver cells. Electron microscopy of insulinimmunogold histochemistry in adult human liver cells treated with (A, B)or without (C) Ad-CMV-PDX-1 and growth factors. Arrows, immunogoldparticles concentrated in secretory granules which appear in PDX-1treated liver cells.

FIG. 20B is a bar chart showing the results of quantitative RT-PCR(Real-Time) gene expression analyses performed using specific Taqmanprobes for the specific endocrine secretory granule molecule markersSecretogrannin2 (SCG2) and Secretory Granule Neuroendocrine 1 (SGNE1),normalized to β-actin gene expression within the same cells, inAd-CMV-PDX-1 and GF treated or untreated adult liver cells. Human isletsserve as a positive control. Results indicate fold of increase (FOI)compared to that of untreated control liver cells (n≧12 in eachexperiment).

FIG. 21A is a bar chart showing the results of Quantitative RT-PCR(Real-Time) gene expression analyses performed using specific Taqmanprobes for the regulatory proteins Glucokinase, Glut-2 and ProhormoneConvertase 2 (PC2), normalized to β-actin gene expression within thesame cells, in Ad-CMV-PDX-1 and GF treated or control liver cells. Humanislets serve as a positive control. Results indicate fold of increase(FOI) compared to (untreated) control liver cells (n≧10 in eachexperiment).

FIG. 21B is a graph depicting insulin secretion in a time course (15-180minutes) at 2 mM (o) or 25 mM (.) glucose concentrations in PDX-1 and GFtreated adult human liver cells.

FIG. 21C is a graph depicting C-peptide secretion in a time course(15-180 minutes) at 2 mM (o) or 25 mM (.) glucose concentrations inPDX-1 and GF treated adult human liver cells.

FIG. 21D is a graph depicting C-peptide dose response secretion at 0-25mM Glucose (.) or 2-DOG (.) concentrations. The arrow and dotted lineindicate exchanging the 25 mM Glucose treatment into 2 mM Glucose.(b-d): n=6 in 3 different experiments.

FIG. 22A is a graph showing PDX-1 treated liver cells correcthyperglycemia in SCID-NOD mice. Diabetic SCID-NOD mice were implantedwith adult human liver cells treated with (.; n=9) or without (□; n=5)Ad-CMV-PDX-1 and growth factors under the kidney capsule. Glucose levelsat the indicated days after transplantation are presented in mg %.Asterisks denote a significant difference (p<0.01) between the glucoselevels of the Ad-CMV-PDX-1 treated implanted mice and the control cellimplanted mice.

FIG. 22B are a series of photographs showing immunohistochemistrystaining for Pdx-1 (A) and insulin (B) in the kidney capsule sections,10 days after implantation of Ad-CMV-PDX-1 treated adult liver cells.Insulin staining of the same SCID-NOD mouse pancreas (C).

FIG. 22C is a bar chart showing Human C-peptide levels in serum of theimplanted and control mice were measured by ELISA before theimplantation experiment (0 days, pre-treated; grey bar) and at the endof the experiment (10 days, treated; black bar). Asterisks (*) denote asignificant difference (p<0.01) between human C-peptide serum levels ofAd-CMV-PDX-1 treated cell implanted mice and the same diabetic miceprior to human cells implantation.

FIG. 23 is a bar chart showing ectopic expression of rat-PDX-1 inducesthe expression of the endogenous human PDX-1 in liver cells. Adultprimary liver cells were treated by Ad-CMV-PDX-1 supplemented withgrowth factors and analyzed for endogenous human-PDX-1 gene expression.Results indicate fold of increase (FOI) compared to untreated controlliver cells.

FIG. 24A is a chart depicting blood glucose levels in diabetic CAD-NODmice treated with Ad-CMV-PDX-1. Control untreated or b-gal treated mice▴, PDX-1 treated mice(.)

FIG. 24B is a bar chart depicting body weight diabetic CAD-NOD micetreated with Ad-CMV-PDX-1. Control untreated or b-gal treated mice ▴,PDX-1 treated mice(.)

FIG. 24C is a series of photographs showing glycogen storage inAd-CMV-PDX-1 treated diabetic mice. C1, treated mice; C2 non-treatedmice; C3 pre-diabetic NOD mice.

FIG. 25 are a series of photographs depicting immunohistochemicalstaining of pancreas and liver in CAD-NOD mice. A, CAD-NOD micenegatively stained for insulin; B, control nondiabetic mice; C insulinin liver of PDX-1 treated mice; D, liver form non-treated diabetic mice.

FIG. 26A is a bar chart depicting serum insulin levels of Ad-CMV-PDX-1treated CAD-NOD mice.

FIG. 26B is a bar chart depicting hepatic insulin levels of Ad-CMV-PDX-1treated CAD-NOD mice.

FIG. 27 is a graph depicting glucose tolerance of Ad-CMV-PDX-1 treatedCAD-NOD mice.

FIG. 28 is a photograph of RT-PCR and amplified products are resolved onagarose gel showing ectopic PDX-1 induced gene expression in CAD-NODmouse livers.

FIG. 29 is a scatter graph depicting PDX-1 induction of change ofexpression in about 500 genes, as analyzed by DNA microarray chipanalysis of human liver cells treated by PDX-1 or by control.

FIG. 30A is a table showing PDX-1 repression of C/EBPβ gene expressionin liver pancreas, human liver cells-control, human liver cells treatedby Ad-CMV-hInsulin, and human liver cells treated by PDX-1 by DNAmicroarray analysis.

FIG. 30B is a bar chart showing PDX-1 repression of C/EBPβ geneexpression by Quantitative RT-PCR analysis.

FIG. 30C is a photograph of a Western Blot showing PDX-1 repression ofC/EBPβ intracellular protein levels.

FIG. 31A is a photograph of a Western Blot showing PDX-1 repression ofhepatic proteins, in mature (left) and Fetal (right) human hepatocytes,and the induction of dedifferentiation of adult human liver cells.

FIG. 31B is a bar chart showing PDX-1 repression of hepatic geneexpression and protein production.

FIG. 31C is a bar chart showing PDX-1 repression of hepatic geneexpression and protein production.

FIG. 31D is a bar chart showing PDX-1 induces dedifferation of matureliver cells as evident by the induction of embryonic markers.

FIG. 31E is a bar chart showing PDX-1 induces dedifferation of matureliver cells as evident by the induction of embryonic markers.

FIG. 32A is a photograph of a RT-PCR and amplified products are resolvedon agarose gel showing ectopic PDX-1 induces pancreatic genes andpancreatic transcription factor expression in human liver cells invitro.

FIG. 32B is a bar chart showing ectopic PDX-1 induces endogenous, humanPDX-1 expression in human liver cells in vitro.

FIG. 32C is a bar chart showing ectopic PDX-1 induces beta2 expressionin human liver) cells in vitro.

FIG. 32D is a bar chart showing ectopic PDX-1 induces Isl-1 expressionin human liver cells in vitro.

FIG. 32E is a bar chart showing ectopic PDX-1 induces Nkx6.1 expressionin human liver cells in vitro.

FIG. 32F is a bar chart showing ectopic PDX-1 induces Ngn3.1 expressionin human liver cells in vitro.

FIG. 32G is a bar chart showing ectopic PDX-1 induces Nkx2.2 expressionin human liver cells in vitro.

FIG. 33 is a bar chart showing ectopic PDX-1 induced insulin geneexpression if augmented by hepatic growth factor-1.

FIG. 34 is a bar chart showing ectopic PDX-1 induces elastase geneexpression.

FIG. 35 is a Western blot analysis showing that ectopic NeuroD1 inducesthe endogenous Pdx-1 in liver cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery that ectopic expressionof pancreatic and duodenal homobox gene 1 (PDX-1) in liver and skininduces a pancreatic islet cell phenotype in liver and skin cells andresults in the expression, production and processing of pancreatichormones. PDX-1 is also known as IDX-1, IPF-1, STF-1 and IUF-1, all ofwhich are collectively referred to herein as “PDX”. Additionally, theinvention provides methods and pharmaceutical compositions for treatingpancreatic disorders.

Recent advance in the outcome of clinical pancreatic islettransplantation for diabetes suggests that continuous control of bloodglucose levels could be achieved by pancreatic islet cells implantation.However this successful therapeutic method is severely restricted bylimited tissue supply from cadaveric donors, and by the need forlifelong immunosuppression. Islet cell implantation, as a treatment fordiabetic patients will be widely available only when new sources ofislets or β-cells are found. The optimal source of tissue engineered toreplace β-cell function in type I autoimmune diabetes should be easilyisolated, largely expanded, and preferentially resists autoimmuneattack; these cells may potentially reside in extra-pancreatic tissues.

Ectopic expression of PDX-1 in-vivo delivered by first generation E1deleted recombinant adenovirus (Ad-CMV-PDX-1) induced both the endocrineand exocrine pancreatic repertoire of gene expression and the productionand secretion of processed, biologically active insulin. These resultsdemonstrate that PDX-1 ectopically expressed in a mature fullydifferentiated organ, PDX-1 functions as a pancreatic differentiationfactor. Moreover, although PDX-1 was delivered by in-vivo using thefirst generation of recombinant adenoviruses, the expression andproduction of pancreatic hormones persisted more than eight months aftertreatment.

This surprising capacity of liver to serve as a potential source oftissue for generating functional endocrine pancreas was firstdemonstrated by us in mice in-vivo using ectopic PDX-1 gene expression.It was shown that short term expression of PDX-1 transgene in miceinduced a comprehensive, irreversible and functionaltransdifferentiation process (i.e., is converting one mature cellcharacteristics and function into another fully differentiated cell) ina sub-population of liver cells. In addition is was demonstrated thatfreshly isolated, adult as well as fetal human liver cells undercontrolled conditions in in-vitro culture can be induced totransdifferentiate into functional insulin producing tissue. About 50%of the liver cells that expressed the PDX-1 transgene activated theotherwise inactive insulin promoter. Transdifferentiated human livercells produced the hormone, stored it in secretory granules and releasedprocessed insulin in a glucose-regulated manner. Insulin-producing humanliver cells were functional and restored normoglycemia in diabeticimmunodeficient mice and yclophosphamide-acceleratted diabetes inducedin non-obese diabetic mice.

In its various aspects and embodiments, the a invention includesadministering to a subject or contacting a cell with a compound (alsoreferred to herein as a PDX inducer compound) that increases PDXexpression or activity. PDX expression or activity is increased forexample by the compound activating endogenous PDX expression. Thecompound can be, e.g., (i) a PDX, a NeuroD or a betacellulinpolypeptide; (ii) a nucleic acid encoding a PDX, a NeuroD or abetacellulin polypeptide; (iii) a nucleic acid that increases expressionof a nucleic acid that encodes a PDX polypeptide and, and derivatives,fragments, analogs and homologs thereof. A nucleic acid that increaseexpression of a nucleic acid that encodes a PDX polypeptide includes,e.g., promoters, enhancers. The nucleic acid can be either endogenous orexogenous. Optionally, the cell is further contacted with nicotinamide,epidermal growth factor, activin A, hepatic growth factor, exendin,GLP-1 or betacellulin.

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculecan be single-stranded or double-stranded. Preferably, the nucleic acidis a DNA. A nucleic acid that increase expression of a nucleic acid thatencodes a PDX polypeptide includes, e.g., promoters, enhancers. Thenucleic acid can be either endogenous or exogenous.

Suitable sources of nucleic acids encoding PDX include for example thehuman PDX nucleic acid (and the encoded protein sequences) available asGenBank Accession Nos. U35632 and AAA88820, respectively. Other sourcesinclude rat PDX nucleic acid and protein sequences are shown in GenBankAccession No. U35632 and AAA18355, respectively, and are incorporatedherein by reference in their entirety. An addition source includezebrafish PDX nucleic acid and protein sequences are shown in GenBankAccession No. AF036325 and AAC41260, respectively, and are incorporatedherein by reference in their entirety.

The compound can be administered to the subject either directly (i.e.,the subject is directly exposed to the nucleic acid or nucleicacid-containing vector) or indirectly (i.e., cells are first transformedwith the nucleic acid in vitro, then transplanted into the subject). Forexample, in one embodiment mammalian cells are isolated from a subjectand the PDX nucleic acid introduced into the isolated cells in vitro.The cells are reintroduced into a suitable mammalian subject.Preferably, the cell is introduced into an autologous subject. Theroutes of administration of the compound can include e.g., parenteral,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration. In oneembodiment the compound is administered intravenous. Preferably, thecompound is implanted under the kidney capsule or injected into theportal vein.

The cell can be any cell that is capable of producing pancreatichormones, e.g., bone marrow muscle, spleen, kidney, blood, skin,pancreas, or liver. In one embodiment the cell is capable of functioningas a pancreatic islet cell, i.e., store, process and secrete pancreatichormones, preferably insulin upon an extracellular trigger. In anotherembodiment the cell is a hepatocyte, i.e., a liver cell. In additionalembodiments the cell is totipotent or pluripotent. In alternativeembodiments the cell is a hematopoietic stem cell, embryonic stem cellor preferably a hepatic stem cell.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Methods of Inducing Pancreatic Hormone Production

In various aspects, the invention provides methods of inducingpancreatic hormone production in a subject. For example, the method caninclude administering to a subject a compound that increases PDXexpression or activity in an amount sufficient to induce pancreatichormone production.

In another aspect, the method includes providing a cell from a subject,contacting the cell with a compound which increases PDX expression in anamount sufficient to increase pancreatic hormone production andintroducing the cell into a subject. In one embodiment pancreatichormone production occurs in-vitro and in-vivo, upon introducing thecell into the subject. In an alternative embodiment, pancreatic hormoneproduction occurs in-vivo upon introducing the cell in the subject.

The pancreatic hormone can be e.g., insulin, glucogon, somatostatin orislet amyloid polypeptide (IAPP). Insulin can be hepatic insulin orserum insulin. In another embodiment the pancreatic hormone is hepaticinsulin. In an alternative embodiment the pancreatic hormone is seruminsulin (i.e., a fully processed form of insulin capable of promoting,e.g., glucose utilization, carbohydrate, fat and protein metabolism).

In some embodiments the pancreatic hormone is in the “prohormone” form.In other embodiments the pancreatic hormone is in the fully processedbiologically active form of the hormone. In other embodiments thepancreatic hormone is under regulatory control i.e., secretion of thehormone is under nutritional and hormonal control similar toendogenously produced pancreatic hormones. For example, in one aspect ofthe invention the hormone is under the regulatory control of glucose.

The cell population that is exposed to, i.e., contacted with, thecompound can be any number of cells, i.e., one or more cells, and can beprovided in vitro, in vivo, or ex vivo.

Methods of Treating or Preventing Pancreatic Related Disorders

Also included in the invention is a method of treating, i.e., preventingor delaying the onset or alleviating a symptom of pancreatic relateddisorders in a subject. In various aspects the method includesadministering to the subject a compound which modulates the PDXexpression or activity. “Modulates” is meant to include increase ordecrease PDX expression or activity. Preferably, modulation results inalteration of the expression or activity of PDX in a subject to a levelsimilar or identical to a subject not suffering from the pancreaticdisorder. In other aspects the method includes administering to thesubject a compound which induces a non-pancreatic cell with pancreaticislet cell function, e.g., capable of expressing insulin, somatostatinor glucagon. In one embodiment the compound modulates PDX expression oractivity.

The pancreatic disorder can be any disorder associated with thepancreas. For example, the method may be useful in treating pancreatichormone insufficiencies, (e.g., diabetes (Type 1 and Type II),insulinomas, and hyperglycemia. Essentially, any disorder, which isetiologically linked to PDX activity, would be considered susceptible totreatment.

A subject suffering from or at risk of developing diabetes is identifiedby methods known in the art such as determining blood glucose levels.For example, a blood glucose value above 140 mg/dL on at least twooccasions after an overnight fast means a person has diabetes. A personnot suffering from or at risk of developing diabetes is characterized ashaving fasting sugar levels between 70-110 mg/dL.

Symptoms of diabetes include fatigue, nausea, frequent urination,excessive thirst, weight loss, blurred vision, frequent infections andslow healing of wounds or sores, blood pressure consistently at or above140/90, HDL cholesterol less than 35 mg/dL or triglycerides greater than250 mg/dL, hyperglycemia, hypoglycemia, insulin deficiency orresistance. Diabetic or pre-diabetic patients to which the compounds areadministered are identified using diagnostic methods know in the art.

The herein-described PDX modulating compound when used therapeuticallyare referred to herein as “Therapeutics”. Methods of administration ofTherapeutics include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The Therapeutics of the present invention maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically-active agents.Administration can be systemic or local. In addition, it may beadvantageous to administer the Therapeutic into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter attached to a reservoir (e.g., an Ommayareservoir). Pulmonary administration may also be employed by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. It mayalso be desirable to administer the Therapeutic locally to the area inneed of treatment; this may be achieved by, for example, and not by wayof limitation, local infusion during surgery, topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant. Various delivery systems are known and can be usedto administer a Therapeutic of the present invention including, e.g.:(i) encapsulation in liposomes, microparticles, microcapsules; (ii)recombinant cells capable of expressing the Therapeutic; (iii)receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987. J Biol Chem262:4429-4432); (iv) construction of a Therapeutic nucleic acid as partof a retroviral, adenoviral or other vector, and the like. In oneembodiment of the present invention, the Therapeutic may be delivered ina vesicle, in particular a liposome. In a liposome, the protein of thepresent invention is combined, in addition to other pharmaceuticallyacceptable carriers, with amphipathic agents such as lipids which existin aggregated form as micelles, insoluble monolayers, liquid crystals,or lamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028;and U.S. Pat. No. 4,737,323, all of which are incorporated herein byreference. In yet another embodiment, the Therapeutic can be deliveredin a controlled release system including, e.g.: a delivery pump (See,e.g., Saudek, et al., 1989. New Engl J Med 321:574 and a semi-permeablepolymeric material (See, e.g., Howard, et al., 1989. J Neurosurg71:105). Additionally, the controlled release system can be placed inproximity of the therapeutic target (e.g., the brain), thus requiringonly a fraction of the systemic dose. See, e.g., Goodson, In: MedicalApplications of Controlled Release 1984. (CRC Press, Boca Raton, Fla.).

In a specific embodiment of the present invention, where the Therapeuticis a nucleic acid encoding a protein, the Therapeutic nucleic acid maybe administered in vivo to promote expression of its encoded protein, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular (e.g., by use of aretroviral vector, by direct injection, by use of microparticlebombardment, by coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See, e.g.,Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868), and thelike. Alternatively, a nucleic acid Therapeutic can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination or remain episomal.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and may be determined bystandard clinical techniques by those of average skill within the art.In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theoverall seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Ultimately, the attending physician will decide theamount of protein of the present invention with which to treat eachindividual patient. Initially, the attending physician will administerlow doses of protein of the present invention and observe the patient'sresponse. Larger doses of protein of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. However,suitable dosage ranges for intravenous administration of theTherapeutics of the present invention are generally about 20-500micrograms (μg) of active compound per kilogram (Kg) body weight.Suitable dosage ranges for intranasal administration are generally about0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Suppositories generally contain active ingredient inthe range of 0.5% to 10% by weight; oral formulations preferably contain10% to 95% active ingredient.

The duration of intravenous therapy using the Therapeutic of the presentinvention will vary, depending on the severity of the disease beingtreated and the condition and potential idiosyncratic response of eachindividual patient. It is contemplated that the duration of eachapplication of the protein of the present invention will be in the rangeof 12 to 24 hours of continuous intravenous administration. Ultimatelythe attending physician will decide on the appropriate duration ofintravenous therapy using the pharmaceutical composition of the presentinvention.

Cells may also be cultured ex vivo in the presence of therapeutic agentsor proteins of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Methods of Inducing Islet Cell Phenotype and Function

The invention also includes a method of inducing or enhancing a one ormore pancreatic islet cell phenotypes in a cell. In one embodiment thepancreatic cell phenotype is induced in a non-islet cell type. Forexample, a non-pancreatic cell is converted (i.e., transdifferentiated)to a pancreatic cell by contacting a cell with a PDX-1 inducer compound.The cell is contacted with a PDX inducer in an amount to induce theexpression of endogenous PDX-1, an embryonic marker, insulin, glucogon,or somatostatin in the non-pancreatic cell. Alternatively the cell iscontacted with a PDX inducer in an amount to repress the expression ofC/EBPβ, albumin or ADH-1 in the non-pancreatic cell. The method includescontacting a cell with a compound that modulates islet cell specifictranscription factors, e.g., PDX-1, beta 2, ISL-2, Nkx6.1, Ngn3.1, orNKx2.2. in an amount sufficient to induce or enhance the pancreaticislet cell phenotype, e.g., beta, alpha and delta islet cells.Preferably, the compound increases PDX expression (e.g. endogenous PDX-1expression), production or activity. Preferably the method induces apancreatic islet β-cell phenotype.

By “pancreatic islet cell phenotype” is meant that the cell displayingone or more characteristics typical of pancreatic islet cells, i.e.hormone production, processing, storage in secretory granules,nutritionally and hormonally regulated secretion or characteristic isletcell gene expression profile. The pancreatic islet cell phenotype can bedetermined for example by measuring pancreatic hormone production, e.g.,insulin, somatostatin or glucagon. Hormone production can be determinedby methods known in the art, e.g. immunoassay, western blot, receptorbinding assays or functionally by the ability to amelioratehyperglycemia upon implantation in a diabetic host.

The cell can be any cell that is capable of expressing a pancreaticislet cell phenotype, e.g., muscle, bone marrow, spleen, kidney, skin,pancreas, or liver. In one embodiment the cell is capable of functioningas a pancreatic islet cell, i.e., store, process and secrete pancreatichormones, preferably insulin upon an extracellular trigger. In anotherembodiment the cell is a hepatocyte, i.e., a liver cell. The cell is amature cell, i.e., a differentiated cell. In additional embodiments thecell is totipotent or pluripotent. In alternative embodiments the cellis a hematopoietic stem cell, embryonic stem cell or preferably ahepatic stem cell.

The cell population that is exposed to, i.e., contacted with, thecompound can be any number of cells, i.e., one or more cells, and can beprovided in vitro, in vivo, or ex vivo.

Methods of Inducing a Pancreatic Islet Gene Expression Profile

The invention also includes a method of inducing or enhancing apancreatic islet gene expression profile in a subject or a cell. By“pancreatic gene expression profile” is meant to include one or moregenes that are normally transcriptionally silent in non-endocrinetissues, e.g., a pancreatic transcription factor an endocrine gene, oran exocrine gene. For example, expression of PC1/3, insulin, glucagon,somatostatin or endogenous PDX-1. The method includes administering to asubject a compound that increases PDX expression or activity in anamount sufficient to induce a pancreatic islet or endocrine geneexpression profile. In one embodiment the method induces PC1/3 geneexpression in a subject.

Induction of the pancreatic gene expression profile can be detectedusing techniques well known to one of ordinary skill in the art. Forexample, pancreatic hormone RNA sequences can be detected in, e.g.,northern blot hybridization analyses, amplification-based detectionmethods such as reverse-transcription based polymerase chain reaction orsystemic detection by microarray chip analysis. Alternatively,expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene. In a specific embodimentPC1/3 gene or protein expression can be determined by its activity inprocessing prohormones to their active mature form. Such methods arewell known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes, or HPLC of the processedprohormones.

Methods of Identifying Genes the Expression of which is Modulated by Pdx

The invention also includes a method of identifying nucleic acids theexpression of which modulated by PDX. The method includes measuring theexpression of one or more nucleic acids in a test cell populationexposed to a compound that modulates PDX activity or expression.Expression of the nucleic acid sequences in the test cell population isthen compared to the expression of the nucleic acid sequences in areference cell population, which is a cell population that has not beenexposed to the compound, or, in some embodiments, a cell populationexposed the compound. Comparison can be performed on test and referencesamples measured concurrently or at temporally distinct times. Anexample of the latter is the use of compiled expression information,e.g., a sequence database, which assembles information about expressionlevels of known sequences following administration of various agents.For example, alteration of expression levels following administration ofcompound can be compared to the expression changes observed in thenucleic acid sequences following administration of a control agent, sucha PDX nucleic acid.

An alteration in expression of the nucleic acid sequence in the testcell population compared to the expression of the nucleic acid sequencein the reference cell population that has not been exposed to thecompound indicates expression of the nucleic acid is modulated by PDX.

The test cell can be taken from any tissue capable of being modulated byPDX, e.g., pancreas, liver, spleen, or kidney. In one embodiment thecell is from a non-endocrine tissue. Preferably, the cell is livertissue.

Preferably, cells in the reference cell population are derived from atissue type as similar as possible to test cell, e.g., liver tissue. Insome embodiments, the control cell is derived from the same subject asthe test cell, e.g., from a region proximal to the region of origin ofthe test cell. In other embodiments, the control cell population isderived from a database of molecular information derived from cells forwhich the assayed parameter or condition is known.

Expression of the nucleic acids can be measured at the RNA level usingany method known in the art. For example, northern hybridizationanalysis using probes which specifically recognize one or more of thesesequences can be used to determine gene expression. Alternatively,expression can be measured using reverse-transcription-based PCR assays.Expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene products. Such methodsare well known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes.

When alterations in gene expression are associated with geneamplification or deletion, sequence comparisons in test and referencepopulations can be made by comparing relative amounts of the examinedDNA sequences in the test and reference cell populations.

The invention also includes PDX modulated nucleic acids identifiedaccording to this screening method, and a pharmaceutical compositioncomprising the PDX modulated nucleic acids so identified.

PDX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PDX protein, orderivatives, fragments, analogs or homologs thereof. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a linear or circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, lentivirus, adenoviruses and adeno-associated viruses),which serve equivalent functions. Additionally, some viral vectors arecapable of targeting a particular cells type either specifically ornon-specifically.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PDX proteins, mutant forms ofPDX, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PDX in prokaryotic or eukaryotic cells. For example, PDXcan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PDX expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J6:229-234), pMFa (Kurjanand Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, PDX can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to PDX mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., “Antisense RNA asa molecular tool for genetic analysis,” Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. Additionally, hostcells could be modulated once expressing PDX, and may either maintain orloose original characteristics.

A host cell can be any prokaryotic or eukaryotic cell. For example, PDXprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Alternatively, a host cell can be a prematuremammalian cell, i.e., pluripotent stem cell. A host cell can also bederived from other human tissue. Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation, transduction, infection or transfectiontechniques. As used herein, the terms “transformation” “transduction”,“infection” and “transfection” are intended to refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. In addition transfection can be mediated by atransfection agent. By “transfection agent” is meant to include anycompound that mediates incorporation of DNA in the host cell, e.g.,liposome. Suitable methods for transforming or transfecting host cellscan be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORYMANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratorymanuals.

Transfection may be “stable” (i.e. integration of the foreign DNA intothe host genome) or “transient” (i.e., DNA is episomally expressed inthe host cells).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome theremainder of the DNA remains episomal In order to identify and selectthese integrants, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Various selectable markers includethose that confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acid encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding PDX orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die). In another embodiment the cells modulated byPDX or the transfected cells are identified by the induction ofexpression of a endogenous reporter gene. In a specific embodiment, thepromoter is the insulin promoter driving the expression of greenfluorescent protein (GFP).

In one embodiment the PDX nucleic acid is present in a viral vector. Inanother embodiment the PDX nucleic acid is encapsulated in a virus. Insome embodiments the virus preferably infects pluripotent cells ofvarious tissue type, e.g. hematopoietic stem, cells, neuronal stemcells, hepatic stem cells or embryonic stem cells, preferably the virusis hepatotropic. By “hepatotropic” it is meant that the virus has thecapacity to preferably target the cells of the liver either specificallyor non-specifically. In further embodiments the virus is a modulatedhepatitis virus, SV-40, or Epstein-Bar virus. In yet another embodiment,the virus is an adenovirus.

Gene Therapy

In one aspect of the invention a nucleic acid or nucleic acids encodinga PDX polypeptide, or functional derivatives thereof, are administeredby way of gene therapy. Gene therapy refers to therapy that is performedby the administration of a specific nucleic acid to a subject. In thisaspect of the invention, the nucleic acid produces its encodedpeptide(s), which then serve to exert a therapeutic effect by modulatingfunction of an aforementioned disease or disorder. e.g., diabetes. Anyof the methodologies relating to gene therapy available within the artmay be used in the practice of the present invention. See e.g.,Goldspiel, et al., 1993. Clin Pharm 12: 488-505.

In a preferred embodiment, the therapeutic comprises a nucleic acid thatis part of an expression vector expressing any one or more of theaforementioned PDX polypeptides, or fragments, derivatives or analogsthereof, within a suitable host. In a specific embodiment, such anucleic acid possesses a promoter that is operably-linked to codingregion(s) of a PDX polypeptide. The promoter may be inducible orconstitutive, and, optionally, tissue-specific. The promoter may be,e.g., viral or mammalian in origin. In another specific embodiment, anucleic acid molecule is used in which coding sequences (and any otherdesired sequences) are flanked by regions that promote homologousrecombination at a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935. In yet anotherembodiment the nucleic acid that is delivered remains episomal andinduces an endogenous and otherwise silent gene.

Delivery of the therapeutic nucleic acid into a patient may be eitherdirect (i.e., the patient is directly exposed to the nucleic acid ornucleic acid-containing vector) or indirect (i.e., cells are firstcontacted with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In a specific embodiment of the present invention, anucleic acid is directly administered in vivo, where it is expressed toproduce the encoded product. This may be accomplished by any of numerousmethods known in the art including, but not limited to, constructingsaid nucleic acid as part of an appropriate nucleic acid expressionvector and administering the same in a manner such that it becomesintracellular (e.g., by infection using a defective or attenuatedretroviral or other viral vector; see U.S. Pat. No. 4,980,286); directlyinjecting naked DNA; using microparticle bombardment (e.g., a “GeneGun®; Biolistic, DuPont); coating said nucleic acids with lipids; usingassociated cell-surface receptors/transfecting agents; encapsulating inliposomes, microparticles, or microcapsules; administering it in linkageto a peptide that is known to enter the nucleus; or by administering itin linkage to a ligand predisposed to receptor-mediated endocytosis(see, e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can beused to “target” cell types that specifically express the receptors ofinterest, etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a gene into cells in in vitro tissueculture by such methods as electroporation, lipofection, calciumphosphate-mediated transfection, viral infection, or the like.Generally, the methodology of transfer includes the concomitant transferof a selectable marker to the cells. The cells are then placed underselection pressure (e.g., antibiotic resistance) so as to facilitate theisolation of those cells that have taken up, and are expressing, thetransferred gene. Those cells are then delivered to a patient. In aspecific embodiment, prior to the in vivo administration of theresulting recombinant cell, the nucleic acid is introduced into a cellby any method known within the art including, but not limited to:transfection, electroporation, microinjection, infection with a viral orbacteriophage vector containing the nucleic acid sequences of interest,cell fusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, spheroplast fusion, and similar methodologies that ensure thatthe necessary developmental and physiological functions of the recipientcells are not disrupted by the transfer. See e.g., Loeffler and Behr,1993. Meth Enzymol 217: 599-618. The chosen technique should provide forthe stable transfer of the nucleic acid to the cell, such that thenucleic acid is expressible by the cell. Preferably, said transferrednucleic acid is heritable and expressible by the cell progeny. In analternative embodiment, the transferred nucleic acid remains episomaland induces the expression of the otherwise silent endogenous nucleicacid.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, but not limited to, injection of epithelialcells (e.g., subcutaneously), application of recombinant skin cells as askin graft onto the patient, and intravenous injection of recombinantblood cells (e.g., hematopoietic stem or progenitor cells) or livercells. The total amount of cells that are envisioned for use depend uponthe desired effect, patient state, and the like, and may be determinedby one skilled within the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

DNA for gene therapy can be administered to patients parenterally, e.g.,intravenously, subcutaneously, intramuscularly, and intraperitoneally.DNA or an inducing agent is administered in a pharmaceuticallyacceptable carrier, i.e., a biologically compatible vehicle which issuitable for administration to an animal e.g., physiological saline. Atherapeutically effective amount is an amount which is capable ofproducing a medically desirable result, e.g., an increase or decrease ofa PDX or gene product in a treated animal. Such an amount can bedetermined by one of ordinary skill in the art. As is well known in themedical arts, dosage for any given patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently. Dosagesmay vary, but a preferred dosage for intravenous administration of DNAis approximately 10⁶ to 10²² copies of the DNA molecule. For example theDNA is administers at approximately 2×10¹² virions per Kg.

Pharmaceutical Compositions

The compounds, e.g., PDX polypeptides, nucleic acid encoding PDXpolypeptides, or a nucleic acid that increases expression of a nucleicacid that encodes ad PDX polypeptide. (also referred to herein as“active compounds”) of the invention, and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, or protein, and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a PDX polypeptide or PDX encoding nucleic acid) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the bather to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 General Methods

The following general methods were used to perform the experimentsdescribed herein.

Recombinant Adenoviruses

AdCMVPDX-1 was constructed as described in by R. Seijffers et al.Endocrinology 140:1133 (1999). It contains the STF-1 cDNA, the rathomolog of PDX-1 ligated into BamH1 site of pACCMVpLpA vector.

AdCMVβ-gal, contains the nuclear localization signal forβ-galactosidase.

AdCMV-hIns, contains the human insulin cDNA under the control of theheterologous cytomegalovirus promoter.

AdRIP-1-hIns, contains the human insulin cDNA under the control of therat insulin promoter-1 (RIP-1). RIP-1, is 410 bases of the 5′ flankingDNA region of the rat insulin-1 gene.

All first-generation recombinant adenoviruses were constructed accordingto Becker et al.³⁹. The gene of interest was ligated into thepACCMV.pLpA plasmid, followed by co-transfection with the adenovirusplasmid pJM17 and followed by harvesting the recombinant virions fromHEK-293 cells. Ad-CMV-PDX-1 carries the rat homologue of PDX-1 whileAd-CMV-hIns carries the human insulin cDNA, distal to a CMV promoter⁴⁰.Ad-RIP-GFP-CMV-PDX-1 is a bi-functional recombinant adenovirus that wasprepared by insertion of 410 nucleotides of the 5′-flanking region ofthe rat insulin-1 gene (supplied by Dr. Larry Moss) in place of theviral CMV promoter in the pACCMV.pLpA plasmid to drive GFP geneexpression (HindIII/PstI), followed by NotI/NotI insert containingCMV-mPDX-1 (mouse homologue of PDX-1) from pACCMV-PDX-1.pLpA plasmid⁴⁰.The specific function of the recombinant virus was analyzed ininsulinoma cell lines compared to non-β cell lines. Preparation of viralstocks was performed as previous described⁴⁰.

Cell Culture

The mouse pancreatic derived cell line β-TC-1, α-TC-1 and the ratpancreatic cell line RIN1046-38 were cultured according to previouslypublished conditions (21,22).

Animals and Recombinant Adenoviruses

Mice were housed in an air-conditioned environment, under a 12-hourlight/dark cycle, and handled according to institutional animal welfareregulation. Balb/c mice (8-9 weeks old, 24-27 gr.). were treated by1-5×10¹⁰ moi of the indicated recombinant adenoviruses by systemicinjection into the tail vein (in a volume of 200-300 μl physiologicalsaline). Blood was drawn from the tail, for determination of glucoseconcentration (Accutrend® GC, Boehringer Mannheim, Mannheim, Germany).Liver was harvested for immunohistochemical staining (fixed in 4%formaldehyde and embedded in paraffin), for analysis of gene expression(total RNA), and for determination of pancreatic hormone content inliver. For the last two analyses, hepatic specimens were immediatelyfrozen in liquid nitrogen, and stored at −70° C.

Male NOD/LtJ and NOD/Scid mice were maintained under pathogen-freeconditions in the Animal Breeding Center of the Weizmann Institute ofScience. Experiments were carried out under the supervision andguidelines of the Animal Welfare Committee. The mice were 1 month old atthe start of the experiments.

Human Liver Cells

Adult human liver tissues were obtained from 8 different livertransplantation surgeries from 4-10 years old children, and two overforty years old individuals.

Fetal human livers were obtained from 4 different deliberate abortionsof 20-22 weeks gestation. Both adult and fetal liver tissues were usedwith approval from the Committee on Clinical Investigations(institutional review board).

Cell Harvest and Culture Conditions

Isolation of human hepatocytes was performed as previously described⁴¹.In short: Liver samples were irrigated at cold Hank's Balanced Saltsolution (HBSS), cut into thin slices (1-2 mm thick), and digested by0.03% Collagenase type I (Worthington Biochemical Corp.) for 20 minutesat 37° C. Dissociated cells were collected, washed twice in HBSS+EGTA (5mM) and collected by centrifugation at 500×g for 5 minutes at 4° C. Thecells were resuspended in Dulbecco's minimal essential medium (1 gr/Lglucose) supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin and 250 ng/ml amphotericin B (Biological Industries,Israel). Cell viability and number were determined and cells were platedon fibronectin (3 μg/cm², Biological Industries, Israel) pre-coatedplates at 1-2×10⁵ cells/ml. The medium was changed daily during thefirst three days in order to remove non-adherent cells. Confluentcultures were split 1:3 using 0.05% trypsin-EDTA solution. The cellswere kept at 37° in a humidified atmosphere of 5% CO₂ and 95% air.

Viral Infections and Growth Factors Treatment

Liver cells were cultured in Dulbecco's minimal essential medium (1 gr/Lglucose) with or without growth factors as indicated separately (EGF 20ng/ml, Cytolab Ltd.; Nicotinamide 10 mM, Sigma), and were infected byrecombinant adenoviruses (500 moi) for 5 days. The recombinantadenoviruses used in this study: Ad-RIP-GFP-CMV-PDX-1, Ad-CMV-PDX-1,Ad-CMV-hIns⁴⁰, Ad-CMV-GFP (Clontech, BD Biosciences).

RNA Isolation and RT-PCR Analysis.

Total RNA was isolated from frozen tissues using Tri-Reagent (MolecularResearch Center, Ohio). RNA samples were treated by 10 units of RQ1RNase-free DNaseI (Promega), for 60 minutes. cDNA was prepared byreverse transcription (Native AMV Reverse Transcriptase, Chimerx), using4 μg DNA-free total RNA and 0.5 μg oligo(dT)₁₅. PCR was performed usingT3 Thermocycler (Biometra, Gottingen, Germany) and products wereseparated on 1.8% agarose gels and visualized with ethidium bromide. Thesequence of the primers used for PCR and reactions conditions were aslisted in table 1 and in table 3. Note that in order to discriminatebetween expression of the endogenous mouse PDX-1 and the ectopic rathomologue, two sets of specific oligonucleotide primers were designed(see table 1 and table 3).

Real Time PCR

RT-PCR was performed on the LightCycler (Roche Applied Science,Mannheim, Germany), using SYBR-Green I dye.

Amplification conditions included initial denaturation at 95° C. 10minutes, followed by 55 cycles for both mouse and rat PDX-1, or 30cycles for β-actin. For both PDX-1 homologues, each cycle includeddenaturation at 95° C. for 15 seconds, annealing at 59° C. and extensionat 72° C. for 15 seconds. Annealing for β-actin was performed at 56° C.for 10 seconds. The fluorescent signal was monitored 5 seconds aftereach cycle at 90° C. for β-actin, 87° C. for mouse PDX-1 and 88° C. forrat PDX-1. Melting curve program was carried out at 68° C. for 40seconds, to analyze the specificity of the generated products.

The RT-PCR for mouse PDX-1 was performed 3 times and for rat PDX-1 2times.

Both rat and mouse PDX-1 levels were normalized to the respectiveβ-actin mRNA levels, in the same samples.

Alternatively, quantitative real-time RT-PCR, was performed using ABIPrism 7000 sequence Detection system (Applied Biosystems).

The TaqMan™ fluorogenic probes, Assay-On-Demand (Applied Biosystems)used in this study were: Human β-actin Hs99999903_m1; Human InsulinHs00355773_m1; Human Glucagon Hs00174967_m1; Human SomatostatinHs00356144_m1; Human PDX-1 Hs00173014_m1; Human Glut-2 Hs00165775_m1;Human Glucokinase Hs00175951_m1; Human PC2 Hs00159922_m1; Human SCG2Hs00185761_m1; Human SGNE1 Hs00161638_m1.

Amplification conditions included initiation at 50° C. for 2 minutes,denaturation at 95° C. for 10 minutes, followed by 40 cycles, each cycleincluded denaturation at 95° C. for 15 seconds and annealing at 60° C.for 1 minute, using the TaqMan PCR Master Mix (Applied Biosystems).Relative quantitative analysis was according to ABI prism 7000 SDSsoftware, using the 2̂(−ΔCt) equation. The mRNA levels were corrected forhuman β-actin mRNA.

Pancreatic Hormones Immunohistochemistry.

Slides of 4 μm paraffin-embedded sections were deparaffinized, incubatedin 3% H₂O₂, and were incubated in blocking solution (for both Insulinand Glucagon detection), using the commercially available Histomouse™-SPKit (Zymed laboratories, South San Francisco, Calif.). Sections werethen incubated for 1 h at 37° C. with monoclonal antibodies againsthuman insulin and against human glucagon (Sigma), both at a dilution of1:200. Slides were exposed to the secondary biotinylated IgG for 30minutes at room temperature and then incubated in strepavidin-peroxidasefollowed by a chromogen peroxide solution. A control using onlysecondary without primary antibodies followed by strepavidin-peroxidaseand a chromogen peroxide solution was performed to rule out possiblebackground of the system.

Radioimmunoassay (RIA) for Pancreatic Hormones.

Pancreas and livers were isolated, immediately frozen in liquidnitrogen, and stored at −70° C. Frozen tissues were homogenized in 0.18NHCl/35% ethanol. The homogenates were extracted overnight at 4° C. withcontinuous stirring, and the supernatants were lyophilized. Samples weredissolved in 0.8 ml RIA Assay Buffer, supplemented by a cocktail ofprotease inhibitors (Sigma). Hepatic insulin and glucagon levels weredetermined using rat radioimmunoassay (RIA, catalog no. SRI-13K andGL-32K, Linco, Mo., USA, and Coat-A-Count, DPC; CA, USA). Somatostatinconcentrations were determined by RIA (Euro-diagnostica, Sweden).Hepatic content of pancreatic hormones was normalized to the weight ofthe extracted tissue.

Determination of Hepatic Function.

Serum biochemistry profile consisting of albumin, AST (Aspartateaminotransferase), ALT (Alanine aminotransferase) and total billirubinwas determined using Olympus AU 2700 Apparatus (Olympus, Germany) inserum samples.

Insulin and C-Peptide Detection

Insulin and C-peptide secretion and content from primary adult livercells were measured by static incubation of 48 hours after 3 days ofinitial viral and growth factors treatment. Insulin secretion into themedia was measured by RIA using the Ultra Sensitive Human Insulin RIAkit (Linco Research) and C-peptide secretion was measured by HumanC-Peptide RIA kit (Linco Research).

Insulin content was measured after homogenizing the cell pellet in 0.18N HCl, 35% ethanol. The homogenates were extracted overnight at 4° C.with continuous stirring, and the supernatants were lyophilized. Sampleswere dissolved in 0.5 ml PBS containing 0.2% BSA and Protease Inhibitorycocktail (Sigma). 100 μl sample were used for the RIA. Insulin contentwas normalized to total cellular protein, measured by the Bio-RadProtein Assay kit.

Glucose Challenge Assay

Adult liver cells were treated with Ad-CMV-PDX-1 and growth factors for5 days. The cells were plated in 6-well plates at 10⁵ cells per well.

For time course analysis the cells were preincubated for 2 hours inKrebs-Ringer buffer (KRB) containing 0.1% BSA, followed by incubationfor the indicated period in media containing 2 mM or 25 mM glucose. Ateach time point media samples were analyzed for insulin (Ultra SensitiveHuman Insulin RIA kit—Linco Research) and C-peptide secretion (HumanC-Peptide RIA kit—Linco Research).

Glucose dose response; Cells were preincubated for 2 hours with KRBcontaining 0.1% BSA, washed and challenged thereafter with increasingconcentrations of D-Glucose or 2-deoxy-Glucose (0-25 mM) for 2 hours. Atthe end of the incubation period at 25 mM glucose, the cells were washedwith KRB and incubated for additional 2 hours in 2 mM glucose containingmedia.

Electron Microscopy

Liver cells were fixed in 2.5% Gluteraldehyde, osmificated, dehydratedwith a graded series of ethanol and propylene oxide, and embedded inAraladite solution (Polyscience Inc.). Ultra-thin sections were cut inan ultramicrotome, stained with 2% uranyl acetate and Reynolds' leadcitrate solution. For post-embedding immunogold reactions, 50-90 nmliver sections were put on nickel grids. The grids were incubated withantibody against insulin (guinea-pig polyclonal; 7.8 μg/ml, Dako) atroom temperature for O.N and then incubated with immunogold-conjugatedantibody against guinea-pig IgG (15-nm gold; diluted 1:40, Dako) for 1.5hours at room temperature. The sections were observed under an electronmicroscope (Jeol 1200EX2).

Cell Transplantation

Five-weeks-old nonobese diabetic severe combined immunodeficient(SCID-NOD) male mice (Weizmann Institute, Israel) were renderedhyperglycemic by intra-peritoneal injection of streptozotocin (Sigma) at180 mg/kg of body weight. When blood glucose reached levels of about 300mg/dl on two consecutive measurements, mice were transplanted under thekidney capsule, with 3×10⁶ human liver cells pretreated with PDX-1 andgrowth factors for 5 days in 50 μl of Matrigel (Sigma) by using a30-gauge needle. Blood was drawn from the tail twice a week, fordetermination of glucose concentration (Accutrend® GC, Roche AppliedScience). Serum was collected in blood of fed mice for C-peptide andinsulin levels analyses, by using the Ultrasensitive Human C-PeptideELISA kit (Mercodia) with 0% cross reactivity to mouse C-peptide and themouse Insulin ELISA kit (Mercodia) with 0% cross reactivity to humaninsulin, according to the manufacturers' instructions. Kidney andpancreas were harvested for immunohistochemical analysis.

Histology and Staining

Kidney and pancreas were fixed in 4% formaldehyde and embedded inparaffin. Sections of the 5 m in thickness paraffin-embedded tissueswere deparaffinized, incubated in 3% H₂O₂, and then were eithermicrowaved in citrate buffer for antigen retrieval before beingincubated in blocking solution (PDX-1 detection) or immediately exposedto the blocking solution (insulin detection), using Histomouse™-SP Kit(Zymed laboratories). For detection of PDX-1, sections were incubatedovernight at 4° C. with antiserum raised against the N-terminal portionof frog PDX-1 (1:5000, a gift from C.V.E. Wright). For detection ofinsulin, sections were incubated for 1 h at 37° C. with a monoclonalantibody against human insulin (1:100; Sigma). Slides were exposed tothe secondary biotinylated IgG for 30 min and then incubated instreptavidin-peroxidase followed by a chromogen peroxide solution.

Cyclophosphamide-accelerated diabetes (CAD)

Diabetes onset was accelerated as previously described (3) withcyclophosphamide (Sigma, Rehovot, Israel). Male NOD mice received ani.p. injection of 200 mg/kg of cyclophosphamide at the age of 4 weeks.If the mice did not become diabetic within the following 2 weeks, theywere given a second i.p. injection (200 mg/kg) of Cy, and the processwas repeated once more, after a week. Diabetic mice were removed fromthe SPF and housed in an air-conditioned environment, under a 12-hourlight/dark cycle, where they were allowed to acclimatize for 72 h beforea new measurement of glycemia was taken to confirm diabetes.

Hyperglycemia

Blood glucose was measured twice weekly using an Accutrend® GC GlucoseAnalyzed (Boehringer Mannheim, Mannheim, Germany). A mouse wasconsidered diabetic when its blood glucose concentration was higher than300 mg/dl on two consecutive examinations

Virus Injection

Diabetic NOD mice 8-10 weeks old (weighting 20-22 gr.) received 1.5×10¹⁰pfu of the indicated recombinant adenovirus by systemic injection intothe tail vein. The viruses were administered in a volume of 200-300 μlof PBS.

Pancreas Histology

Pancreata and livers were fixed in 4% formaldehyde, embedded inparaffin, cut and stained by standard hematoxylin and eosinImmunohistochemical staining, slides of 4 μm paraffin-embedded sectionswere deparaffinized, blocked and analyzed using the Histomouse™-SP Kit(Zymed laboratories, South San Francisco, Calif.), as described in (21).Sections were incubated with a 1/100 dilution of a monoclonal antibodyto human insulin (Sigma, Rehovot, Israel). Slides were developed usingan anti-mouse IgG biotinylated antibody in combination withstreptavidin-peroxidase followed by a chromogen peroxide solutionControl slides were developed in parallel following the same protocolbut without adding the insulin-specific antibody.

Example 2 Determination of PDX-1 Induced Endogenous Insulin GeneExpression and Activation of Ectopically Co-Delivered Insulin Promoter

To assess the effect of ectopic PDX-1 expression in the liver, maleBalb/c and C57BL/6 mice (11-14 week old) were injected with 2×10⁹ plaqueforming units (in 0.2 ml saline) of AdCMV-PDX-1 recombinant adenovirusinto the tail vein. As controls, mice were similarly injected withAdCMV-β-gal, or AdCMV-hIns and AdRIP-1-hIns recombinant adenoviruses.The animals were housed in an air-conditioned environment, under a12-hour light/dark cycle, on a regular unrestricted diet, and sacrificedone week following virus administration. The liver, spleen, pancreas andkidney were dissected and were immediately frozen in liquid nitrogen,and stored at −70° C. for total RNA isolation.

PDX-1 and insulin gene expression was determined by RT-PCR. Total RNAwas isolated from frozen tissues using RNAzol (CINNA-BIOTEX). RNAsamples were treated by 10 ul of DNase I (Promega). cDNA was prepared byreverse transcription, using 1 μg DNA-free total RNA and 0.5 μgoligo(dT)₁₅. 1.5 μl of RT reaction was amplified using primers and PCRconditions as indicated in Table 1 below. PCR was carried out in aGeneAmp PCR system 2400 (Perkin Elmer), and products were separated on1.7% agarose gel. A separate PCR reaction was carried out for each RNAsample without reverse transcriptase, to ensure that the amplifiedproduct was not due to DNA contamination. The primers were designed todetect the only the ectopic rat PDX-1 expression not the mouse homolog.The primers for nil-2 amplification are located on different exons. Thefirst step of sample denaturation was identical for all amplified genes:94° C. for 1 minute.

Analysis of the total RNA revealed that AdCMV-PDX-1 administrationresulted in PDX-1 expression mainly in liver. Spleen, pancreas andkidney from the same mice tested negative by RT-PCR for the rat homologof PDX-1.

75% (25 of 35) of the mice that tested positive for the ectopic ratPDX-1 message expressed the mI-2 gene whereas 35% of the mice expressedmI-1 gene (FIG. 1). To determine whether this disparity of expressionbetween mI-2 and mI-1 was due the mI-1 promoter being differentiallyeffected by the identity or the levels of transcription factors presentin PDX-1 expressing liver cells, AdRIP-1-hIns recombinant adenovirus wasco-delivered with AdCMV-PDX-1 to mice as described above. Asdemonstrated in FIG. 1, in livers where PDX-1 induced only theexpression of the endogenous mI-2, it also activated the rate insulin-1promoter (RIP-1). This suggests that the different levels of DNAmethylation or distinct chromatin structure could be the cause of thelow efficiency of the activation of the endogenous nil-1 expression byPDX-1 expression in the liver. Furthermore these data demonstrate thecapacity to activate the β-cell specific insulin promoter in liver whenco-delivered with PDX.

The expression of the endogenous mouse insulin and the ectopic humaninsulin genes was not induced by treatment with the same concentrationof the control recombinant adenoviruses AdCMV-β-gal, or AdCMV-hIns andAdRIP-1hIns, respectively (n=20). These results demonstrate that PDX-1is essential and sufficient to induce expression of the endogenousinsulin genes and to activate RIP-1 in an extra-pancreatic tissue.

Example 3 Determination of PDX-1 Induced Somatostatin Gene Expressionand Protein Production In-Vivo

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Somatostatin gene expression was determined by RT-PCR as described inEXAMPLE 2, according to the conditions described in Table 1.

As demonstrated in FIG. 3 livers in mice treated with AdCMV-PDX-1exhibited somatostatin gene expression. Mice treated with AdCMV-PDX-1exhibited positive staining for the somatostatin protein in liver tissueanalyzed by immunochemistry. Mice treated with AdCMV-β-gal did notexpress somatostatin.

Example 4 Determination of PDX-1 Induced Glucagon Gene Expression

Animals were treated with AdCMVPDX-1 recombinant adenovirus as describedin EXAMPLE 2 Glucagon gene expression was determined by RT-PCR asdescribed in EXAMPLE 2, using conditions and primers as described inTable 1.

As demonstrated in FIG. 3 livers in mice treated with AdCMV-PDX-1exhibited glucagon gene expression. Mice treated with AdCMV-β-gal didnot express glucagon.

Example 5 Determination of Prohormone Convertase 1/3 Induced GeneExpression

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Prohormone convertase 1/3 (PC1/3) gene expression was determined byRT-PCR as described in EXAMPLE 2 with the exception that cDNA wasreverse-transcribed using a gene specific oligonucleotide(TCCAGGTGCCTACAG GATTCTCT) (SEQ ID NO:1) instead of oligo (dT)₁₅). Asdemonstrated in FIG. 3 only livers from animals treated with PDX-1exhibited the induction of PC1/3 expression, a member of the Kexinfamily proteases, PC1/3 expression is restricted to endocrine andneuroendocrine cells with regulated secretory pathway. Taken togetherwith the capacity to retain the produced hormones in intracellularcompartments suggests a PDX-1 dependent induction of an endocrinephenotype which includes the induction of a regulated pathway forhormone production, processing, storage and secretion.

TABLE 1 RT-PCR analysis for determination of PDX-1 induced gene-expression.PCR Conditions Primer Sequences PCR Annealing Extension Gene 5′-3′Product ° C. sec ° C. sec Cycles Rat PDX-1 F: CCAGTTTGCAGGCTCGCTGG279 bp 62 60 72 60 31 (ectopic) (SEQ ID NO: 2) R: GCTGCGTATGCACCTCCTGC(SEQ ID NO: 3) Human Insulin F: CTTTGTGAACCAACACCTGTGC 239 bp 63 60 7260 38 (ectopic) (SEQ ID NO: 4) R: GCAGATGCTGGTACAGCATTGT (SEQ ID NO: 5)Mouse Insulin I F: TTGCCCTCTGGGAGCCCAAA 253 bp 62 60 72 60 38(SEQ ID NO: 6) R: CAGATGCTGGTGCAGCACTG (SEQ ID NO: 7) Mouse Insulin IIF: TCTTCCTCTGGGAGTCCCAC 259 bp 62 60 72 60 38 (SEQ ID NO: 8)R: CAGATGCTGGTGCAGCACTG (SEQ ID NO: 9) Mouse β-actinF: ATGGATGACGATATCGCT 500 bp 56 45 72 60 35 (SEQ ID NO: 10)R: ATGAGGTAGTCTGTCAGGT (SEQ ID NO: 11) Mouse PC1/3F: CTGGTTGTCTGGACCTCTGAGTA 361 bp 55 45 72 60 38 (SEQ ID NO: 12)R: CCAACAGCAGAA GTGAGTGTGAC (SEQ ID NO: 13) Mouse PDX-1F: CAAGCTCGCTGGGATCACTGGAGCAG 421 bp 58 45 72 60 38 (endogenous)(SEQ ID NO: 14) R: GATGTGTCTCTCGGTCAAGTTCAACATC (SEQ ID NO: 15)Mouse & Rrat F: CCTGGCTTTGGGCGGTGTCA 165 bp 68 45 72 60 38 somatostatin(SEQ ID NO: 16) R: CTCGGGCTCCAGGGCATCATTC (SEQ ID NO: 17) MouseF: ACCAGCGACTACAGCAAATACCTC 242 bp 60 45 72 60 38 glucagon(SEQ ID NO: 18) R: AGCAATGGCGACTTCTTCTGG (SEQ ID NO: 19) rat insulin-1F: GTGACCAGCTACAATCATAG 370 bp 57 45 72 60 38 (SEQ ID NO: 20)R: AGTTCTCCAGTTGGTAGAGG (SEQ ID NO: 21) Rat β-actinF: CGTAAAGACCTCTATGCCAA 350 bp 57 45 72 60 35 (SEQ ID NO: 22)R: AGCCATGCCAAATGTGTCAT (SEQ ID NO: 23)

TABLE 3  Primer sequences and PCR conditions: CDNA Product AnnealingGene Primer sequences (μl) (bp) ° C. sec Cycles β actinF: ATGGATGACGATATCGCT 1 570 56 45 35 (SEQ ID NO: 26)R: ATGAGGTAGTCTGTCAGGT (SEQ ID NO: 27) Rat F: CCAAAACCGTCGCATGAAGTG 1628 60 60 35 PDX-1* (SEQ ID NO 28) R: CAGCTCGCCTGGTGGCTGT(SEQ ID NO: 29) Mouse  F: CCTTCGGGCCTTAGCGTGTC 3 396 59 90 38 PDX-1**(SEQ ID NO: 30) R: CGCCTGCTGGTCCGTATTG (SEQ ID NO: 31) Insulin IF: TTGCCCTCTGGGAGCCCAAA 1 253 62.6 60 38 (SEQ ID NO: 32)R: CAGATGCTGGTGCAGCACTG (SEQ ID NO: 33) Insulin F: TCTTCCTCTGGGAGTCCCAC1 259 62.6 60 36 II (SEQ ID NO: 34) R: CAGATGCTGGTGCAGCACTG(SEQ ID NO: 35) Somatostatin F: CAGACTCCGTCAGTTTCTGC 3 262 54 90 38(SEQ ID NO: 36) R: ACAGGATGTGAAAGTCTTCCA (SEQ ID NO: 37) GlucagonF: ATCATTCCCAGCTTCCCAGA 2 161 52 60 38 (SEQ ID NO: 38)R: CGGTTCCTCTTGGTGTTCAT (SEQ ID NO: 39) IAPP F: CCACTTGAGAGCTACACCTG 2205 54 60 37 (SEQ ID NO: 40) R: GGATTCCCTATTTGGATCC (SEQ ID NO: 41)Ad-CMV- F: CTCAATGGGAGTTTGTTTTGG 1 569 8 0 26 PDX-1 (SEQ ID NO: 42)(DNA) R: GGGGGATTAGCACTGAACTCT (SEQ ID NO: 43) ElastaseF: GGGCACAAACAGACCATCAC 2 298 5 0 38 (SEQ ID NO: 44)R: GGGATGGGTAAGAAGGTGGT (SEQ ID NO: 45) P48 F: GAAGGTTATCATCTGCCATCG 3211 4 0 38 (SEQ ID NO: 46) R: GGGTGGTTCGTTCTCTATGTT (SEQ ID NO: 47)RT-PCR reaction conditions: denaturation at 94° C. for 1 min; annealingas presented in the table and extension at 72° C. for 1 min. *specificprimer to rat PDX-1, no recognition of mouse PDX-1 **specific primer tomouse PDX-1, no recognition of rat PDX-1

Example 6 PDX-1 Induced Proinsulin Synthesis in Liver S

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Liver, spleen, pancreas and kidney were dissected. Portions of thetissue fixed in 4% formaldehyde and embedded in paraffin forimmunohistochemical staining. The remaining liver and pancreatic tissueswere homogenized in 70% ethanol-0.18N HCl, lyophilized and resuspendedin PBS (phosphate buffered saline) for RIA determination of IRI content.

Immunohistochemistry

Five μm sections of paraffin-embedded tissues were deparaffinized,incubated in 3% H₂O₂, and then, either microwaved in citrate buffer forantigen retrieval prior to incubation in blocking solution (PDX-1detection), or immediately exposed to the blocking solution (insulindetection). (Histomouse™-SP Kit, Zymed laboratories, CA, USA).

PDX-1 detection: sections were incubated overnight at 4° C. withantiserum raised against the N-terminal portion of frog PDX-1.

Insulin detection: sections were incubated for 1 hour at 37° C. with amonoclonal anti-human-insulin (Sigma, St. Louis Mo.).

Slides were exposed to the secondary biotinylated IgG for 30 minutes,incubated in streptavidin-peroxidase followed by chromogen-peroxidesolution.

Immunohistochemical analysis of liver sections from mice treated withPDX-1, revealed expression of the homeoprotein in 30-60% of hepatocytenuclei, with 0.1-1% of the liver cells staining positive for(pro)insulin. Control AdCMVβ-gal treated livers, did not stain positivefor (pro)insulin although β-galactosidase activity was evident in 50% ofthe nuclei. Livers from mice treated by AdCMV-hIns, did not stainpositive for insulin in the hepatic sections, although serum IRI fromthe same mice was three fold increased, as were serum IRI levels inPDX-1 treated mice. The fact that the ectopic expression of PDX-1 butnot of insulin resulted in positive immunostaining for (pro)insulin maysuggest the induction of a cellular modulation which supports insulinretention in a small subpopulation of liver cells, (secretory vesicleswhich belong to the regulated pathway, characteristic to endocrinecells, but not to liver cells), which may have shifted toward a β-cellphenotype.

Radioimmunoassay

To determine whether hepatic insulin mRNA is effectively translated intoprotein, immunoreactive insulin (IRI) content was tested in extractsderived from hepatic tissue by radioimmunoassay (RIA). Livers from PDX-1treated mice that tested positive for insulin gene expression by RT-PCR(FIG. 1) contained about 25 fold more IRI than livers of animals treatedby a control virus (Table 2). Mean IRI levels in extracts derived fromPDX-1 treated livers was 20.7±3.97 μU/mg protein, while in controllivers, IRI was only 0.78±0.25 μU/mg protein. The background level ofinsulin measured in control liver samples possibly represents insulin(of pancreatic origin) bound to its receptors which are abundant in thisorgan. While IRI detected in PDX-1 treated liver extracts was <1% of thelevels detected in pancreatic extracts (Table 2), serum IRI levels inPDX-1 treated mice were almost 3-fold higher compared to controls(323±48.4 vs. 118.2±23.7 μU/ml, respectively (Table 2)), indicating thatinsulin was being synthesized and a large portion of it secreted intothe blood stream. These data indicate that the insulin gene expressioninduced by the molecular manipulation is successfully translated intospecific hepatic production of the pro/hormone.

Immunoreactive insulin detected in PDX-1 treated livers was less than 1%of IRI levels in pancreatic extracts (Table 2). The IRI valuesdetermined by radioimmuno-assay (RIA) in liver extracts mayunder-estimate the actual insulin production in this organ. The antibodywe used for RIA preferentially binds the processed hormone, and has only60% cross-reactivity with proinsulin, which is expected to be presentmainly in hepatocytes and to a much lower extent in pancreas.

Example 7 Blood Glucose and Serum Insulin Levels

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Prior to sacrifice, blood was drawn from the inferior vena cava fordetermination of glucose concentration (Accutrend® GC, BoehringerMannheim, Mannheim, Germany) and insulin levels by radioimmunoassay(Coat-a-count, DPC, Los-Angeles, Calif., USA, using rat insulinstandards, (Linco), the anti-insulin antibody used has only 60%cross-reactivity with human proinsulin).

Mice treated by AdCMV-PDX-1 recombinant adenoviruses were nothypoglycemic, however, their blood glucose levels were significantlylower than of mice treated by AdCMV-β-gal or AdCMV-Luc[197±11.2 vs.228±15.74 mg/dl, respectively (Table 2). Plasma immunoreactive insulinlevels were significantly higher in PDX-1 treated mice compared tocontrols [323±48.4 vs. 118.2±23.7 μU/ml respectively (Table 2).

The three fold increase in serum IRI levels in PDX-1 treated mice,cannot by itself explain the twenty-fivefold increase (Table 2) inhepatic IRI content demonstrated in PDX-1 treated liver extracts. Thus,the increase in hepatic pro/insulin content originates from localproduction.

TABLE 2 Blood glucose and immunoreactive insulin (IRI) levels in serumand liver extracts. Control virus AdCMV-PDX-1 treated mice treated miceBlood glucose, mg/dl   228 ± 15.74 (n = 18) 197 ± 11.2 (n = 40) SerumIRI, μU/ml 118.2 ± 23.7 (n = 14) 323 ± 48.4 (n = 26) Liver extracts IRI 0.78 ± 0.25 (n = 10) 20.7 ± 3.97 (n = 12)  μU/mg protein Pancreasextracts IRI 2627 ± 24 (n = 6)  μU/mg protein

Example 8 HPLC Analysis of Insulin-Related Peptides

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Liver, and pancreas were dissected and homogenized in 70%ethanol-0.18N HCl, lyophilized and resuspended in 0.1 M HCl-0.1% BSA forHPLC analysis.

Insulin-related peptides from the liver and pancreatic extracts wereresolved by reverse-phase HPLC using Lichrospher 100 RP-18 column(Merck, Darmstadt, Germany) and elution conditions as described by Grosset al. One ml fractions were collected into tubes containing 0.1 ml 0.1%BSA in water, dried in a Speed-Vac apparatus and reconstituted in 1 mlRIA buffer (0.1% BSA in PBS) for peptide determination by RIA. Guineapig antiporcine insulin antibodies (Linco, St Charles, Mo.) with eitherrat or human insulin standards were used for determination of mouse orhuman IRI, respectively.

HPLC analysis of hepatic IRI content from PDX-1 treated mice revealed59±7% (n=3) conversion into fully processed mI-1 and mI-2. Incomparison, pancreatic extracts contained 85±5% (n=3) mature insulin(FIG. 2) Whereas, ectopic expression of human insulin (AdCMV-hIns) didnot result in retention of IRI in the liver cells except for one liverin which most of the extracted IRI was immature insulin. This is in linewith previous observations in transfected FAO cells in which noretention of the insulin gene product observed and most of it wassecreted by the constitutive secretory pathway. These data demonstratesthat ectopic PDX-1 expression in liver induces a cellular machinery,characteristic to endocrine tissue capable of processing the inducedprohormone, and is not induced when only proinsulin is ectopicallyexpressed in liver. Thus, inducing an extended n-cell phenotype in livercells by ectopic PDX-1 expression.

Example 9 Biological Activity of Hepatic Pro/Insulin Production

The ability of PDX-1-induced hepatic insulin production to control bloodglucose levels in diabetic mice was studied. C57BL/6 mice were rendereddiabetic (>600 mg/dl) with ketoacidosis, 24 hours after 200 mg/kgintraperitoneal STZ injection. 24-48 hours after STZ injection, micewere treated by either AdCMV-PDX-1 or by AdCMVβ-gal (control)recombinant adenoviruses administered via the tail vein, in salinesolution. As demonstrated in FIG. 4, AdCMV-PDX-1 treated mice, exhibitedgradual decrease in blood glucose levels from about 600 to 200-300 mg/dlstarting two days after recombinant adenoviral treatment. In contrast,in the control AdCMVβ-gal treated mice, hyperglycemia persisted and wasaccompanied by increased rate of mortality, 12 out of 22 tested died,with severe ketoacidosis 1-3 days after adenovirus treatment.Furthermore, both groups lost weight after induction of hyperglycemia,and did not regain it back before mice were sacrificed. In summary, thedata demonstrate that expression of PDX-1 is sufficient to inducemature, biologically active insulin production in liver whichameliorates hyperglycemia in mice bearing ablated β-cell function.

Example 10 In-Vitro Activation of Insulin Promoter by Ectopic PDX-1Expression

PDX-1 activates rat insulin-1 promoter when co-delivered with arecombinant adenovirus AdRip-1hIns in which human insulin expression isdelivered by a rat insulin-1 promoter. (See, EXAMPLE 2 and FIG. 1. PDX-1was shown to be sufficient to activate rat insulin promoter-1 in-vitroin rat liver cells. Primary cultures if mature and fetal hepatocyteswere cultured on collagen-1 covered tissue culture dishes in serum freechemically defined media. Two days after plating cells were treated byeither AdCMV-PDX-1& AdRIP-1hIns or by AdCMV β-gal & AdRIP-1hIns. 48hours after adenoviral treatment, total RNA was extracted and proinsulingenes expression was assessed as described in EXAMPLE 2.

PDX-1 activated the ectopically expressed RIP-hIns (rat insulinpromoter-1, 410 bps of this promoter, driving human insulin, introducedvia recombinant adenovirus), while β-gal did not possess such acapacity. (FIG. 5)

Example 11 In-Vitro Induction of Endogenous Somatostatin Gene Expressionin Hepatocytes

Primary cultures of hepatocytes isolated from fetal (E14-Fisher-344rats) were cultured and treated by recombinant adenoviruses as describedin EXAMPLE 9. Somatostatin gene expression was detected in reversetranscribed total RNA samples as described in EXAMPLE 2, using primersand RT-PCR conditions as described in Table 1.

The data demonstrate that ectopic PDX-1 expression in hepatocytesin-vitro induces the expression of the endogenous, otherwise silentsomatostatin gene expression in hepatocytes, in-vitro (FIG. 6).

Example 12 In-Vitro Induction of Endogenous Insulin Gene Expression inHepatocytes

Primary cultures of fetal (E14-Fisher-344 rats) were cultured andtreated by recombinant adenoviruses as described in EXAMPLE 10. Ratinsulin 1 gene expression was detected in reverse transcribed total RNAsamples as described in EXAMPLE 2, using primers and RT-PCR conditionsas described in Table 1.

The data demonstrate that ectopic PDX-1 expression in primary culture offetal hepatocytes in-vitro induces the expression of the endogenous,otherwise silent insulin gene expression (FIG. 6).

Example 13 Ectopic PDX-1 Expression in Liver Cells Induces anIntracellular Compartment Characteristic of Endocrine and NeuroendocrineCells which Allows the Retention of the Produced Hormones, and itsRegulated Secretion

Mice were treated with either Ad-CMVhIns or AdCMVPDX-1 as described inEXAMPLE 2. Treatment resulted in a three-fold increase serum IRIdemonstrating human insulin production by liver cells (FIG. 1). Cellspositive for the insulin protein by immunocytochemistry were detectedonly in AdCMVPDX-treatment. Moreover, HPLC analysis of liver extractsdetected only trace levels of IRI in liver extracts all of itunprocessed in the Ad-CMVhIns treated mice compared to 25 fold increasein the AdCMVPDX-1 treated mice. Furthermore, 59% of the insulin producedin AdCMVPDX-1 treated mice was processed. In addition, only liverstreated by AdCMVPDX-1 exhibited the induction of the prohormoneprocessing enzyme PC1/3 which is characteristic only to cells capable ofregulated pathway for insulin processing storage and regulatedsecretion. These data demonstrate that PDX induces the regulatedsecretion of insulin in liver cells

Example 14 Identification of Nucleic Acids the Expression of which isModulated by PDX

Nucleic acids modulated by PDX are identified by ectopic PDX expression.Nucleic acids that are not expressed in control treated extra-pancreaticislet tissue, as compared to pancreatic tissue are the nucleic acidsmodulated by PDX. These nucleic acids so identified are used astherapeutic compounds to treat pancreatic associated disorders.

Identification of the target genes is performed by either subtractivelibraries, commercially available microarray Chips (Incyte, orAffimetrix), or membrane hybridizations (CLONTECH. Atlas™ expressionarrays, or Multiple Tissue Northern (MTN®)Blots). RNA isolation fromtreated tissues, its purification, and cDNA probe synthesis is performedaccording to manufacturer instructions.

The genes which are expressed in the PDX treated non-pancreatic islettissue and are also present in pancreatic islets probed membranes orchips, but not in control treated non-pancreatic islet tissue, are thedirect and non-direct PDX target genes, which represent the islet cellscharacteristic profile of gene expression. Discrimination between director indirect is elucidated by candidate target gene promoter analysis byelectromobility shift assay (EMSA) as in FIG. 7, and promoterfootprinting (as described in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Example 15 Inducing Regulated Expression of a Desired EctopicallyExpressed Gene in Host Tissue

This EXAMPLE illustrates the induction of regulated expression of anyreporter, in addition to insulin. When PDX activates the insulinpromoter in non pancreatic islet tissue, and mediates its glucose andgrowth factors sensing ability, than, any additional promoter will besimilarly regulated by glucose and growth factors. Thus, this inventioncan be utilized to nutritionally and hormonally regulate expression ofnumerous secreted/or non secreted factors such as, for example,glucagon, growth hormone, steroid hormones which are driven by theinsulin promoter thus controlling their transcription, and regulatedsecretion, from an otherwise non-endocrine tissue. (FIG. 7.)

Example 16 Identification of PDX Location in the Hierarchy of B or IsletCell Specific Transcription Factors

This EXAMPLE illustrates the identification of the PDX location in thehierarchy of β-cell or islet cell specific transcription factors. Everytranscription factor expressed in pancreatic islets but is not inducedby ectopic PDX-1 expression in liver, could cooperate with PDX for theinduction of a more comprehensive, complete or close to complete β-cellphenotype in non-endocrine-pancreatic tissue, such as liver. Thedetection of induced expression of islet cell specific transcriptionfactors in liver is performed as in EXAMPLE 2, using the appropriateprimers and conditions the example of which is elaborated in Table 1.

An additional method to analyze the activity of transcription factors isperformed by footprinting, and by

Electro-Mobility Shift Assays (EMSA):

Nuclear extracts (3-4 μg of protein) were incubated on ice for 10minutes in DNA binding mixture containing 10% Glycerol, 15 mM Hepes (pH7.9), 150 mM KCl, 5 mM DTT and 0.3 μg of poly dIdC, poly dAdT (SIGMASt-Louis Mo.). After the first incubation, approximately 0.2 ng of theprobe was added for an additional 25 minutes incubation on ice. Thebinding reaction was analyzed on a native 4% polyacrylamide gel.

Oligonucleotides (Probes)

Synthetic double-stranded oligonucleotides are end-labeled with[α³²P]ATP using the Klenow fragment of DNA polymerase. The sequences ofoligonucleotides A3/A4 which is an example for PDX-1 binding site (oneof them) on the insulin promoter 5′GATCTGCC CCTTGTTAATAATCTAATG 3′ (SEQID NO:24). The sequence for A1 (additional PDX-1 binding site on insulinpromoter) is 5′ GATCCGCCCTTAATGGGCCAAACGGCA-3′ (SEQ ID NO:25). Thelabeled oligos are used as probes for electromobility shift assays, asdescribed in FIG. 7. The identity of PDX-1 is double estimated bysupershift using a specific antibody which prevents the PDX-1 binding toits cognate locus on the promoter, or that increases the molecularweight of the complex separated on PAGE (antibody+pdx-1+probe) comparedto that which includes only pdx-1+labeled probe (last two lanes in FIG.7).

Example 17 Ectopic PDX-1 Expression Induces Pancreatic Endocrine andExocrine Markers in the Liver

Ectopic PDX-1 expression in mature liver in-vivo activates a widerepertoire of pancreatic genes. Both endocrine and exocrine markersincluding the exocrine pancreas transcription factor p48 were uniquelyexpressed in response to ectopic PDX-1 expression in liver (FIG. 8).Control treated mice were mostly negative to pancreatic gene expression.While insulin gene expression was induced in close to 100% of micetreated by ectopic PDX-1, it was expressed at very low levels that werenot translated into protein also in 20% of the control treated mice.

In the developing pancreas PDX-1 serves as an early molecular markerthat temporally correlates with pancreatic commitment. This datademonstrates that that PDX-1 recapitulates its role in pancreasorganogenesis, in a mature fully differentiated tissue, such as liver.

Example 18 PDX-1 Triggers a Long-Lasting Process of Liver to PancreasDevelopmental Shift

Insulin, glucagon, and somatostatin gene expression and proteinproduction for six months after the initial, single adenovirus mediatedPDX-1 administration to mice livers in-vivo was assessed.

Eight to nine weeks old mice were treated by systemic injection ofAd-CMV-PDX-1, a recombinant adenovirus that carries the rat PDX-1 gene(STF-1) under the control of the CMV promoter. Pancreatic geneexpression in liver was analyzed in comparison to age-matched controlmice (treated by either Ad-CMV-β-Galactosidase or untreated).

Despite the expected transient PDX-1 expression achieved byadenovirus-mediated delivery of the gene to liver (expression ofrecombinant PDX-1 wanes between 30 and 56 days after viral injection),expression of insulin and somatostatin persisted for 6 months, at boththe mRNA (FIG. 9) and protein levels (FIG. 11). Glucagon gene expressionwas evident during the first four months (FIGS. 9 & 10). Importantly,insulin I and insulin II genes expression was evident in 80-100% ofPDX-1 treated mice even six-eight months after initial PDX-1 treatment.

The temporal differences between insulin and glucagon gene expressioncould reflect a unique phenomenon that characterizes pancreasorganogenesis in mature liver, and suggests a more stabletransconversion toward the β and δ cell phenotype. Glucagon gene is nota direct PDX-1 target gene, and its persistent expression in liversuggests that PDX-1 is functioning as a differentiation factor in thisorgan.

Example 19 Quantitative Analysis of Insulin, Glucagon and SomatostatinHormones Production in PDX-1 Treated Livers

Immunohistochemical analysis (FIG. 10) localizes the insulin producingcells mainly in the proximity of central veins even four to six monthsafter PDX-1 ectopic gene delivery (FIGS. 10A & 10C). Although glucagonpositive cells are also localized in the proximity of central veins(FIG. 10B), immunohistochemical analysis of these two hormones performedon sequential slides suggest that these hormones do not co-localizewithin the same cell. Liver cells present in areas close to the centralvein in liver are known to correspond to mature cells.

Quantitative analysis of hepatic insulin stored in liver of PDX-1treated mice indicates that even four to six months after treatment,hepatic insulin content is about 30-75 ng/g tissue, compared to 1-9 ng/gtissue in age-matched control livers (FIG. 11A). A significant two-foldincrease in hepatic pro/glucagon and somatostatin content was observedup to at least four months after initial Ad-CMV-PDX-1 administration(FIGS. 11 B & C). The substantial differences in hepatic content ofinsulin as compared to that of the other two pancreatic hormonesfollowing PDX-1 treatment may resemble the ratio of these hormones inthe pancreas. Despite hepatic insulin production, serum insulin andglucose levels in PDX-1 treated mice, bearing normal pancreatic functionwere normal throughout the duration of the experiment (insulin: 1.0±0.5vs. 0.9±0.4 ng/ml, and glucagon: 0.16±0.08 vs. 0.12±0.05 ng/ml in PDX-1treated compared to controls, respectively).

The persistent production of pancreatic hormones in liver suggests thatectopic PDX-1 triggers a cascade of events that does not require thecontinuous presence of the PDX-1 transgene.

Example 20 Ectopic PDX-1 Triggers the Expression of the Endogenous,Otherwise Silent PDX-1 Gene in Liver

To evaluate the sustained developmental shift in liver, triggered by thetransient ectopic PDX-1 expression, whether the transgene induces theexpression of otherwise silent pancreatic transcription factors wasanalyzed.

To analyze the induction of the endogenous and otherwise silent PDX-1gene in liver by the ectopic gene, mice were treated by systemicdelivery of recombinant adenovirus that directs expression of the ratPDX-1 homologue, and used specific oligonucleotide primers todistinguish between the ectopic PDX-1 transgene (rat) mRNA (cDNA) andthe endogenous mouse mRNA, by RT-PCR.

PCR analysis of DNA samples isolated from liver of Ad-CMV-PDX-1 treatedmice confirms that the virally encoded transgene disappears between 30and 56 days after adenovirus injection (FIG. 12A).

FIG. 12B demonstrates that the ectopic rat PDX-1 expression parallelsthe observed presence of delivered viral DNA in liver and alsoextinguishes after one month (FIG. 12A). The only homologue of PDX-1expressed in treated livers for the whole duration of the experiment isthe endogenous and otherwise silent mouse homologue (FIG. 12B).Endogenous PDX-1 expression is exclusive to mice that received the ratPDX-1 transgene, and was evident in 75% of ectopic PDX-1 treated mice(21 out of 28 mice), and in none of the 25 control treated liversanalyzed. Using real time PCR the identity and quantitated the relativelevels of mouse versus rat PDX-1 gene expression in liver was analyzedas a function of time after the initial treatment, using identicalconditions (but different primers) and normalized it to β-actin withinthe same samples.

As shown in FIG. 12C, the mRNA encoding the ectopic rat PDX-1 is maximalat 5 days, drops by 85% at day 30 and disappears thereafter. Bycontrast, the endogenous mouse PDX-1 is expressed at substantial levelsfor the whole duration of the experiment. These data taken together,suggest an auto-induction of the endogenous and otherwise silent PDX-1in liver, which suggests a mechanistic explanation for the long lastingmode of liver to pancreas transconversion process.

Example 21 Insulin Produced in Liver in Response to PDX-1 TransgeneExpression is Functional and Prevents STZ-Induced Hyperglycemia

In order to determine whether PDX-1 gene delivery induces long-lastingproduction of biologically active insulin, whether it providesprotection against STZ induced diabetes was analyzed. Eight months afterthe initial Ad-CMV-PDX-1 treatment, mice were treated by 220 mg/kg STZ,and the incidences of hyperglycemia were compared to these in agematched controls. Sixty percent of the control Balb/c mice developedhyperglycemia (6 out of 10), within 3-5 days of STZ injection. Bycontrast, only one out of five PDX-1 treated mice developedhyperglycemia in response to STZ treatment (20%), despite the fact thatthey were analyzed eight months after Ad-CMV-PDX-1 treatment.

Immunohistochemical studies and quantitation of insulin content levelsby RIA revealed that in response to STZ treatment, pancreatic β-cellswere mostly destroyed and pancreatic insulin content in both controldiabetic mice and importantly in PDX-1 treated mice (that remainednormoglycemic) dropped by 95±1%. By contrast, hepatic immunoreactiveinsulin (IRI) content in PDX-1 treated mice was forty fold increasedcompared to control diabetic mice that were not treated by PDX-1 (FIG.13). In healthy mice hepatic insulin is only about 1% of that producedin pancreas However, in response to STZ treatment, hepatic insulinproduction becomes 25.6% of the amount of immunoreactive insulinproduced in STZ treated pancreas of the same mice.

These results demonstrate that the PDX-1 induced developmental shift isboth long lasting and functional, the relatively modest IRI levelscontributed by the liver, suggests that insulin produced in liverprotects against STZ induced hyperglycemia also by efficient regulationof the balance between hepatic glucose production and glucose disposal.Importantly, it also suggests that developmentally shifted cells inliver resist β-cell specific toxins.

Importantly, despite the ongoing local insulin production in liver evensix-eight month after initial viral infection, hepatic functions werenot perturbed (Table 4a). Transient alterations in hepatic functionsoccurred in response to adenovirus administration, however, hepaticfunction returned back to normal levels within one-two months. Moreover,serum amylase levels were not increased at all time points despiteendogenous PDX-1 and pancreatic hormones expression (table 4b). The rateof weight increase of PDX-1 treated mice was similar to that in agematched control mice.

TABLE 4 Hepatic function in Ad-CMV-PDX-1 treated mice ALB AST ALT T. bil(gr/dl) (IU/L) (IU/L) (mg/dl) Control (10) 2.5 ± 0.05   82 ± 5.75 36.6 ±3.2 0.1 PDX 5 days (5) 2.5* 121* 102* 0.1* PDX 20 days (5) 2.5 ± 0.05111 ± 21  73 ± 10 0.1 PDX 60 days (12) 2.5 ± 0.05 101 ± 31  53.6 ± 13.60.1 PDX 120 days (3) 2.5  81.6 ± 29  29 ± 2 0.1 PDX 180 days (3) 2.5 ±0.05 61 ± 7 21 ± 2 0.1 Blood biochemistry measurements (mean ± SEM) ofmice after PDX administration. ALB: albumin; AST: aspartateaminotransferase; ALT: alanine aminotransferase; T. bil: totalbilirubin. Data are mean ± SEM; *pooled samples, numbers of miceanalyzed are in parenthesis.

TABLE 4b Serum Amylase levels in PDX-1 treated mice. Time afteradenoviruses Amylase(IU/L) Administration Control PDX-1  5 days 1850 (3)1709 (2)  60 days 1909 (2) 1876 (2) 120 days 2240 (3) 1744 (7) 180 days1978 (4) 2477 (4) 240 days 2298 (6) 2343 (4) 280 days 2634 (3) 2570 (4)

Example 22 PDX-1 and Growth Factors Induced Transdifferentiation inPrimary Culture of Human Keratinocytes

Cell Culture

Keratinocyte cultures were initiated from small biopsy specimens (2-4cm²) of split-thickness skin. After overnight (ON) incubation intrypsin-EDTA the epidermis was separated and epithelium disaggregated intrypsin-EDTA to form single cells suspension. The cell suspension wascultured in Keratinocyte Medium (Nature 265: 421-4, 1977), and the cellsuspension was attached to falcon culture plates and used at passages2-5. When cells reached 70% confluency, they were treated by theindicated treatments described below for 48-96 hours.

Gene Expression

Gene expression analyses were performed using Taqman real time PCR(ABI).

Cell Treatment K1: EGF + KGF + NIC + K2: EGF + KGF + NIC + PDX-1(100moi) PDX-1(10 moi) K3: EGF + KGF K4: EGF + KGF + RGCI

At all treatments: EGF, KGF are 20 ng/ml; NIC: 10 ng/ml

Controls

The control cells were treated with a non-relevant, Ad-CMV-Hinsulin,recombinant adenovirus that carried the expression of the human insulingene under the control of the CMV promoter. RGCI is a bifunctionalrecombinant adenovirus construct—Ad-CMV-PDX-1-RIP-GFP that identifiedcells that had undergone PDX-1 mediated transdifferentiation towardinsulin gene expression. PDX-1 expression in this virus was driven byCMV promoter, whereas GFP expression was driven by the tissue specificpromoter for insulin (RIP).

Results

In the treatments K1-K4, the endogenous otherwise silent pancreaticgenes were expressed in keratinocytes. Interestingly, glucagon geneexpression was induced by low levels of PDX-1 (K4) and importantly, byEGF+KGF treatment alone, with no need for ectopic PDX-1. (FIG. 15).

Example 23 PDX-1 Activates the Insulin Promoter in Human Liver Cells

Human liver cells were isolated from both adult and fetal tissues. Thecells exhibited a heterogeneous phenotype and proliferated efficientlyin culture for up to 20 passages. It was analyzed whether human cellsisolated from adult fully differentiated liver without any priorselection undergo a process of developmental re-direction towardspancreatic phenotype in response to ectopic PDX-1 expression. The firstindication of pancreatic characteristics is the activation of theinsulin promoter, which otherwise is inactive in liver. Cells weretreated by the bi-functional recombinant adenovirus:Ad-RIP-GFP-CMV-PDX-1 which carries the expression of PDX-1 under thecontrol of the heterologous CMV promoter, while the insulin promotercontrols GFP expression (FIG. 17 a), thereby Pdx-1 ‘responding’ cellswere identified by green fluorescence (FIG. 17 b). The total capacity ofadult human liver cells to be infected by recombinant adenovirus wasexamined using Ad-CMV-GFP infection; 40±7% of adult liver cells atpassages 1-6 expressed green fluorescence in response to Ad-CMV-GFPinfection. Surprisingly, about half of these cells (23±3.5%) respondedto ectopic PDX-1 expression by activation of the pancreatic promoter(FIG. 17 b), as was also determined by FACS analysis (data notpresented). In order to determine whether the partial response isinfluenced and limited by the differentiation state of the adult livercells, PDX-1 capacity to induce a developmental redirection of fetalhuman liver cells, being less differentiated and possibly contain morepluripotent cells than adult human liver cells was analyzed. Indeed,27±7.8% of fetal human liver cells in culture (isolated from 22 weeksgestation) responded to ectopic PDX-1 expression by activation of thepancreatic promoter, while their response to Ad-CMV-GFP transduction wassimilar to that of cells isolated from adult liver (FIG. 17 c). Thismodest increase in number of responding cells, may suggest that thedifferentiation state of the cells plays only a limited role in thedevelopmental shift process induced by PDX-1.

Three important observations emerge from the primary culture of humanliver cells. First, both adult and fetal cells, when cultured in-vitro,proliferate efficiently for up to six months and are capable ofactivating the insulin promoter in response to PDX-1 treatment. However,their infection and ‘transdifferentiation’ capacities decrease with theincrease in passage number (FIGS. 17 c&d). Second, although fetal humanhepatic tissue may consist a larger number of pluripotent cells thancells isolated from adult liver, they may possess similar capacities toundergo a transdifferentiation process towards pancreas (FIG. 17 c).Third, the capacity to activate the insulin promoter in human livercells that ectopically express PDX-1 does not occur in a rare populationof cells, since half of the cells capable of being infected byrecombinant adenovirus also activated the ectopic insulin promoter in aPDX-1 dependent manner at low passages in culture (FIG. 17 d).

Example 24 Soluble Factors Promote PDX-1 Induced Liver to PancreasTransdifferentiation

A better indication for the extent of the transdifferentiation processis to analyze the induction of the endogenous otherwise silentpancreatic genes expression in PDX-1 treated liver cells.

The expression of the three major pancreatic hormones genes expressionwas induced by PDX-1 more than two orders of magnitude, compared tocontrol untreated liver cells (FIG. 18).

Nicotinamide and epidermal growth factor (EGF) are known to promotepancreatic endocrine differentiation of undifferentiated pancreaticcells, including that of embryonic pancreatic organ culture.Interestingly, when PDX-1 treatment was supplemented with nicotinamideand EGF (collectively called GF), pancreatic hormones gene expressionwas dramatically increased. Insulin gene expression in primary cultureof adult liver cells was seven orders of magnitude increased compared tothat in control untreated liver cells. Neither nicotinamide nor EGFalone or their combination exhibited PDX-1 independent effect onpancreatic gene expression in liver cells. These data suggest that PDX-1is necessary to the process of liver to pancreas transdifferentiation,while the GF possess a synergistic effect on the process without beingsufficient to independently induce it. Importantly, fetal and adulthuman liver cells exhibited similar levels of pancreatic gene expressionin response to ectopic PDX-1 expression and GF treatment, as depicted byreal-time PCR quantification for insulin gene expression in bothcultures (FIG. 18 b).

Culturing liver cells in the presence of GF for couple of weeks prior toPDX-1 treatment did not result in increased insulin gene expressioncompared to cells that were simultaneously treated by PDX-1 and GF. Incontrast, excluding GF from pretreated cultures resulted in insulin geneexpression at levels similar to that of PDX-1 alone. Taken together itis suggested that the promoting effect GF have on PDX-1 inducedtransdifferentiation is not due to inducing the proliferation of a raresubpopulation of cells susceptible to the process, but rather theycontribute in an yet unknown fashion to possibly augment theintra-cellular signal transduction leading to the PDX-1 induced process.Similar multiplicity of infection of Ad-CMV-hIns, a recombinantadenovirus that carries the constitutive ectopic expression of humanproinsulin cDNA under the control of the CMV promoter resulted ininsulin gene expression at levels comparable to these in PDX-1 and GFtreated cells (without inducing glucagon and somatostatin geneexpression, as under PDX-1 treatment), Taking in consideration that thenumber of cells that express insulin when treated by Ad-CMV-hIns istwice the number of cells that express the PDX-1 induced endogenousinsulin gene (since only up to 23% of the cells may undergo atransdifferentiation process, while 40% of the cells express the ectopichuman insulin gene, see FIGS. 17 c and 18 c), suggests that the(endogenous) insulin promoter in PDX-1 treated liver cells is as activeand potent as the heterologous CMV promoter.

Example 25 PDX-1 Endows Adult Human Liver Cells with EndocrineCharacteristics

In order to analyze whether once transdifferentiated, adult human livercells gain endocrine cells characteristics, these cells potential tostore and process, the PDX-1 induced insulin was analyzed. FIG. 19demonstrates the insulin and C-peptide secretion and the insulin contentin PDX-1 treated cells. PDX-1 treatment alone resulted in 34.5±4.5 foldincrease in immunoreactive insulin (IRI) content, 38.7±8.7 fold increasein IRI secretion and 7.5±2.1 fold increase in C-peptide secretion.Supplementing the culture media by GF substantially augmented the PDX-1effect on the process; IRI content raised to 91.3±20.3 fold increase,its secretion was 74.5±33.3 fold increased and C-peptide secretion was33.9±14.6 fold increased compared to untreated liver cells. The effectof PDX-1 on the process was compared to that of ectopic expression ofhuman proinsulin (using the Ad-CMV-hIns recombinant adenovirus). Whilemost of the IRI produced in cells treated by Ad-CMV-hIns was released,much of the IRI in PDX-1 treated cells is retained within the cells. Themodest secretion of C-peptide upon Ad-CMV-hIns treatment could beattributed to pre-existing endopeptidases such as furin in liver cells.Importantly, the induction of prohormone convertase 2 was evident onlyupon PDX-1 treatment but not in Ad-CMV-hIns treated liver cells (FIG. 21a).

Electron microscopic analysis of immunogold histochemistry usingantibodies against insulin revealed that the insulin is stored insecretory granules (FIG. 20 a). These granules did not contain acharacteristic dense core as in intact pancreatic islets in-vivo, butresembled these present in the β-cell lines that may contain a lowerlevel of insulin storage. The endocrine phenotype was associated withthe specific induction of neuroendocrine vesicles specific geneexpression. Specific expression of SCG-2 (Secretogranin-2) and SGNE1(Secretory granule neuroendocrine-1, FIG. 21 b) was observed only inPDX-1 treated cells but not upon Ad-CMV-hIns treatment.

These data taken together suggest that adult human liver cells treatedby PDX-1 and soluble factors undergo a wide and efficienttransdifferentiation process into pancreatic hormones producing cells,that resembles many features characteristic to pancreatic endocrinecells.

Example 26 Glucose Sensing Ability of Transdifferentiated Adult HumanLiver Cells

Glucose sensing ability and the coupling between glucose sensing andinsulin secretion is the hallmark of pancreatic β-cell function.

It was demonstrated that PDX-1 induced transdifferentiated liver cellsexpress GLUT-2 and glucokinase (GK, FIG. 21 a) genes and secrete insulinin a glucose regulated manner. Exposure of PDX-1 treated adult humanliver cells to 25 mM glucose results in an immediate and profoundincrease in insulin secretion (FIG. 21 b). Time course analysis ofglucose stimulated insulin (FIG. 21 b) and C-peptide (FIG. 21 c)secretion, revealed similar bi-phasic dynamic characteristics as that inpancreatic β-cells, with an immediate and sharp first peak followed by aprolonged second peak of secretion. Once the glucose trigger wasremoved, insulin secretion immediately decreased (FIG. 21 d). Thedecrease in extra-cellular insulin levels 60-90 minutes after theinitial glucose trigger (FIG. 21 b), was more profound than in C-peptidesecretion (FIG. 21 c) and may represent extensive uptake of the secretedinsulin by liver cells in the heterogeneous culture. Glucose doseresponse reveals a shift to the right compared to normal pancreaticβ-cells, since maximal C-peptide secretion occurs at 25 mM glucose (FIG.21 d) compared to maximal insulin secretion at 8-16 mM in normalpancreatic islets. Importantly, the coupling between the glucose sensingability to insulin secretion occurs in transdifferentiated liver cellsin the same mode as in normal pancreatic β-cells: glucose should bemetabolized in order to exert its effect on insulin secretion. Anon-metabolizable glucose analog; 2-deoxy-glucose (2-DOG), did nottrigger C-peptide secretion in transdifferentiated liver cells (FIG. 21d). As expected, ectopic expression of human insulin driven by aconstitutive promoter (Ad-CMV-hIns) did not result in glucose regulatedsecretion of the prohormone. These data indicate that the glucosesensing ability and its coupling to insulin secretion are a consequenceof the transdifferentiation process.

Example 27 Transdifferentiated Adult Human Liver Cells AmeliorateHyperglycemia in Diabetic Mice

To determine the ability of transdifferentiated adult human liver cellsto replace β-cell function, the cells were transplanted intoimmunodeficient, SCID-NOD mice, which were rendered diabetic by STZtreatment. FIG. 22 demonstrates that whereas control treated miceremained hyperglycemic, mice implanted by adult human liver cellstreated by PDX-1, exhibited a gradual and significant decrease in bloodglucose levels. Immunohistochemical analysis reveals that while thesemice pancreata were empty of insulin, human liver cells implanted underthe kidney capsule stained positive for PDX-1 and insulin (FIG. 18 b).Human C-peptide could be detected in the serum of STZ-treated mice thatwere implanted by PDX-1 treated human liver cells. Human C-peptidelevels were significantly 6-7-fold increased and averaged 0.26±0.03ng/ml, compared to 0.04±0.02 ng/ml (P<0.01), in both normal SCID-NOD andthe STZ-treated control mice (FIG. 22 c). Serum mouse insulin levels inhuman cells implanted mice remained unchanged and were significantlylower (0.16±0.03 ng/ml) than in control normoglycemic SCID-NOD mice(0.45±0.03 ng/ml). Taken together; these findings indicate that thehyperglycemia in the implanted mice was normalized by human insulin thatwas secreted from the transdifferentiated human liver cells. Theseresults establish the capacity of PDX-1 treated transdifferentiatedadult human liver cells to function as surrogate β-cells in-vivo.

Example 28 PDX-1 Induced Liver to Pancreas Transdifferentiation RevertsHyperglycemia in Cad-Nod Mice

To test the effect of PDX-1 induced liver-to-pancreastransdifferentiation on overt autoimmune diabetes, diabetic NOD micewere treated with Ad-CMV-PDX-1 or Ad-Rip-βGal as previously described. Agroup of mice was left untreated as a control. Blood glucose levels andbody weight, to asses the regulation of glucose metabolism weremonitored. Non-treated mice and mice treated with Ad-Rip-βGal remainedhyperglycemic, lost weight, and died within the first two weeksfollowing treatment (FIG. 24). In contrast, 65% (20 out of 34 mice) ofthe mice treated with Ad-CMV-PDX-1 became normoglycemic within the first5 days following treatment. However, this normoglycemia was transient insome mice. While 38% of the mice treated with PDX1 (13/34) remainednormoglycemic 1 month after treatment, the other 20% (7/34) treated withAd-CMV-PDX-1 became hyperglycemic 10-14 days after treatment (FIG. 24a), while maintaining stable body weight for the whole duration of theexperiment (FIG. 24 b).

These data suggest that the process of liver to pancreastransdifferentiation induced by PDX-1 reverses autoimmune diabetes (e.g.Type 1).

Example 29 Synthesis and Regulation of Insulin in Ad-CMV-Pdx-1-TreatedDiabetic Mice

Analysis by immunohistochemistry of pancreatic and hepatic insulinexpression, revealed the presence of insulin-producing cells in theliver of mice treated with Ad-CMV-PDX-1, but not in their pancreas (FIG.25). The hepatic insulin producing-cells were located close to centralveins, as previously described. In addition, hepatic insulin content,and serum insulin levels were significantly higher inAd-CMV-PDX-1-treated mice than in the control groups (FIG. 26). Thus,Ad-CMV-PDX-1 induced the expression of insulin in the liver and itssecretion to the blood.

To analyze the regulation of hepatic insulin release by blood glucose,glucose tolerance tests in 5 diabetic mice 2-3 weeks after they becamenormoglycemic following Ad-CMV-PDX-1 therapy were conducted. Ascontrols, diabetic mice treated with Ad-CMV-βGal and healthy BALB/c micewere. No difference was seen in the rate of glucose clearance betweenhealthy BALB/c and the Ad-CMV-PDX-1-treated mice, indicating thatinsulin secretion by liver transdifferentiated cells is indeed regulatedby glucose (FIG. 27). In contrast, diabetic Ad-CMV-βGal-treated micefailed to show glucose clearance and remained hyperglycemic throughoutthe test. These data taken together indicate that PDX-1 inducedfunctional liver to pancreas transdifferentiation occurs also in themouse model of autoimmune. The PDX-1 induced normoglycemia wasassociated with the induction of insulin production in liver and itsrelease in a glucose regulated manner.

Example 30 PDX-1 Induced Developmental Shift of Bone Marrow Cells

To ability of ectopic PDX-1 to induce a developmental shift of bonemarrow cells to functional pancreatic cells is determined as follows.AC133+ cells isolated from fresh and frozen human BM at purities greaterthan 90% are expanded in either (a) 24-well tissue culture plasticplates or (b) Teflon bags, in the continuous presence of IL-6, TPO,Flt-3 ligand, and SCF with or without TEPA for three weeks. Theprogenitor cell composition and potential are examined at the end of thetreatment time and after long-term incubation in culture.

In order to optimize the infection and experimental conditions, thebifunctional adenovirus (AdRIP-GFP-CMV-PDX-1) is used to infect thecultures. Expression of the GFP-reporter gene as function of time afterinfection at 2-10³ MOI (number of viral particle divided by the numberof cells in culture, MOI, multiplicity of infection). is followed

The responding cells (that activate the insulin promoter and thereforeexpress GFP) are sorted and cultured separately from non-respondingcells for further analysis. A separated culture of control BM cells(either treated or untreated by TEPA) are treated by AdCMV-insulin ascontrol for insulin production without transdifferentiation.

Gene expression is followed by RT-PCR. The levels of genes expressionwith time after the induction of the developmental shift, are analyzedby real time PCR using Real time PCR (ABI, USA) in a quantitative assay.The expression of pancreatic hormones, insulin, glucagon, somatostatinand prohormone convertases, and specific pancreatic transcriptionfactors: BETA2, Isl-1, Nkx6.1, Pax4, and Pax6 and the endogenous PDX-1are analyzed.

Hormones production is detected by immunohistochemistry using specificantibodies; guinea pig anti porcine-insulin; rabbit anti human-glucagon;rabbit anti human-somatostatin (all from DAKO A/S, Glostrup, Denmark).Production and secretion of insulin and glucagon is analyzed bycommercially available RIA specific kits: Sensitive human insulin andC-peptide RIA kits and glucagon RIA kit, (Linco research ICN, Mo., USA).Glucose dose response and time course of pro/insulin synthesis andconversion to mature biological active insulin is resolved byreversed-phase HPLC as described (2), and by analyzing secretion ofC-peptide into the culture medium using a specific human c-peptide RIAkit (Linco). The intracellular insulin content in the different glucoseconcentrations are analyzed.

The dynamics of the response of insulin secretion to glucose, in PDX-1induced BM cells is analyzed in vitro and compared to that of ectopicinsulin expression (AdCMV-hIns). Cells are incubated in differentconcentration of glucose in 6-well dishes, and a dose response and timecourse of IRI secretion to the medium will be measured byradioimmunoassay (RIA). Increased insulin secretion by forskolin/IBMXwill indicate the induction of a regulated pathway for protein secretionin modulated liver cells. Sensitivity to glucose, but not to 2-DOG orL-glucose, indicates specific coupling of insulin secretion pathway tonutrient metabolism in the “modulated” liver cells

Proliferation and transdifferentiated bone marrow cells capacity tocontrol blood glucose levels and revert diabetes in diabetic SCID miceis analyzed to fully determine their capacity to mimic pancreatic b-cellfunction.

Example 31 DNA Microarray Chip Analysis of Pdx-1 Treated Human LiverCells

As shown in Figure DNA microarray analysis of PDX-1 treated human livercells revealed over 500 genes that were either up-regulated ifdownregualted compared to control cells. Genes that were modulated inresponse to PDX-1 treatment includes pancreatic transcription factors(See, Table 5) and catalase and hepatic dismutase polypeptides (See,Table 6)

TABLE 5 Pancreatic Transcription Factors Induced in PDX-1 Treated HumanLiver Cells Panc/ PDX/ Liver Pancreas Cont INS PDX-1 Liver C IPF1 1.87.1 2 1.3 3357 0.7   9.1 PAX6 14.4 1761.7 6.4 6.5 6.9 7.3   0.4 NKX2-212.5 836 1.6 0.7 1.4 6.5   0.1 NEUROD1 13.7 634.9 8.3 12.7 3.3 5.4 −1  ISL1 3 595.1 3 10.1 11.1 6.4  2* MEIS1 36.3 442.8 188.8 217.7 252.7 3.5  0.5 MEIS2 73.6 396.4 120.9 147.8 116.1 2.4 −0.2 STAT1 240.3 255.5637.7 1362.4 1458.9 0 1  PBX3 78.2 201.7 133.7 170.5 182.7 1.4   0.3FOXA2 423.7 195 1.4 2.2 1.5 −0.3   0.1 PBX2 111.8 156.4 18.4 29.9 55.80.5 1  GATA6 88.6 106.5 219.2 151.3 69.5 0 −1   FOXA1 88 50.3 20.3 9.819 −1.1 0  GATA4 48.6 37.8 86.4 95.8 59.3 −0.9 −0.4 GLI2 16.3 33.1 4551.4 67.3 0   0.5 GLI3 52.5 16.4 80.5 94.3 98.8 −1.7 0  *IS1-1upregulated to the same levels by Ad-CMV-INS Pancreatic genesupregulated by PDX-1 treatment are identified in Table 7.

TABLE 7 Pancreatic Genes Upregulated by PDX-1 treatment Liver pancreasont INS PDX-1 Panc/Liver PDX/Cont secretogranin II SCG2 1.4 1735.3 0.913 11.1 2.9 (chromogranin C) transmembrane 4 NET-6 250.3 1429.7 4.8 94.2.5 2.3 superfamily member tetraspan NET-6 regenerating islet- REG1A21.8 3857.5 .9 20. 7.6 2.1 derived 1 alpha (pancreatic stone protein,pancreatic thread protein) secretagogin, EF-hand SCGN 26.8 2080.8 0.129. 5.4 2.1 calcium binding protein secretory granule, SGNE1 47.1 4793.110 1 376. 6.3 1.8 neuroendocrine protein 1 (7B2 protein) regulator ofG-protein RGS5 72.7 3585.3 2.8 78. 5.4 1.8 signalling 5 tumor-associatedTACSTD1 2.7 678.7 .2 11. 9 1.7 calcium signal transducer 1thrombospondin 4 THBS4 11.4 629 5.6 37. 5.3 1.4 p21 (CDKN1A)- PAK3 48.8742.3 2 27. 3.9 1.4 activated kinase 3 protease, serine, 1 PRSS1 28.12878.8 .7 3. 7.2 1.3 (trypsin 1) chromogranin A CHGA 25.6 4120.8 8.4 36.5.6 1.1 (parathyroid secretory protein 1) protocadherin 17 PCDH17 83.5768.3 1.1 41. 2.4 1.1 serine protease SPINK1 39.6 7719.1 6. 6.3 1inhibitor, Kazal type 1 enolase 2, (gamma, ENO2 4.3 256.4 4.4 31 117.3 61.8 neuronal) protein tyrosine PTPRN 4.8 565.2 5.7 20.2 59.3 5.9 1.1phosphatase, receptor type, N amphiphysin (Stiff- AMPH 6.3 126.2 .3 20.639.4 4.1 1.5 Man syndrome with breast cancer 128 kDa autoantigen)neurobeachin NBEA 17.5 168.5 2.8 33.5 28.4 3.1 2.1 protein kinase PKIA27 177.6 50.9 68.6 148.5 2.5 1.6 (cAMP-dependent, catalytic) inhibitoralpha chromosome 3 open C3orf4 32.3 131.8 93.1 152.7 240.1 2.3 1.2reading frame 4 endothelin receptor EDNRB 37.1 280.3 94.4 101.9 593.82.3 2.6 type B chromosome 1 open C1orf9 48.6 138.9 42.2 62.7 92.3 2.31.3 reading frame 9 synuclein, alpha (non SNCA 33.5 146.6 21.2 34.4 1102.3 2.3 A4 component of amyloid precursor) regulator of G-protein RGS287.4 553.4 58.6 122.8 335 2.3 2.2 signalling 2, 24 kDa inhibin, beta AINHBA 49.3 13.6 174 470 1169 −2 2.7 (activin A, activin AB alphapolypeptide) PDX-1 treatment in liver cultures down-regulated theexpression of a variety of pancreatic genes. These pancreatic genesdown-regulated by PDX-1 treatment are identified in Table 8.

TABLE 8 Pancreatic Genes Down-regulated by PDX-1 treatment liverpancreas cont INS PDX-1 Panc/Liver PDX/C alcohol deh  

  ADH1B 1906.1 120.9 579.4 316.6 6.4 −4.2 −6.4 G protein-c  

  GPR65 27 9.8 20.1 16.7 2.9 −1.3 −2.9 D-aspartate  

  DDO 56.1 8.9 27.2 18.3 2.8 −2 −2.7 retinol dehy  

  RDH5 152.7 17.1 46.4 30.9 8.6 −3.2 −2.3 retinoic ac  

  RARRES3 186 72.3 245.9 176.8 68.3 −1.7 −2.2 vascular ce  

VCAM1 243.9 104.4 733.3 603.9 154.4 −2.2 −2.1 macrophag  

  HML2 74.2 18.9 19 15 2.9 −2 −1.8 protein pho  

  PPP1R3C 639.8 180.6 1054.6 944 329.8 −1.8 −1.8 sulfotransfe  

  SULT1A1 2415.8 381 673.8 386.7 177.8 −2.5 −1.7 v-maf muso  

  MAF 191.7 58.6 67.3 31.1 12.7 −2 −1.7 biliverdin re  

  BLVRB 597.4 163.6 351.7 248.6 104.3 −1.8 −1.7 apolipoprot  

  APOL1 242 15.1 348.1 323.9 110.8 −3.4 −1.6 p8 protein  

  P8 584.2 195.2 1641.2 1322.3 425.5 −1.5 −1.5 phosphory  

  PYGL 262.1 39.8 152.6 145.1 60.4 −2.8 −1.3 Fc fragmen  

  FCGRT 828.7 256 239.6 170.2 99.2 −2.2 −1.2 sulfotransfe  

  SULT1A3 855.5 342.3 272.5 215.2 128 −1.5 −1.2 aldo-keto r  

  AKR1C3 1308.5 96.2 246.6 202.4 132.2 −3.6 −1.1 Sulfotransf  

  — 940.8 199.3 259 212.2 130.9 −2.1 −1.1 related RA  

  RRAS2 228 67.1 451.5 415.2 183.6 −2.1 −1.1 serine (or c  

  SERPINF1 2340.7 592.9 529.7 434.1 331.3 −2.1 −1 retinoid X  

  RXRA 665.9 377.6 158.1 107.4 87.4 −1.1 −1 compleme  

C1R 3610.9 290.3 1692 1407.8 971.9 −3.8 −1 calcium reg  

  CARHSP1 612.5 54.1 275.3 203.4 151.6 −2.6 −1 follistatin  

  FST 135.4 38.4 98.5 117.5 51.6 −1.7 −1

indicates data missing or illegible when filed

The following abbreviations are used in the second column of Table 8:ADH1B=alcohol dehydrogenase 1B (class 1); GPR65=G protein-coupledreceptor 65; DDO=D-aspartate oxidase; RDH5=retinol dehydrogenase 5(11-cis and 9-cis); RARRES3=retinoic acid receptor responder (tazaroteneinduced) 3; VCAM=Vascular cell adhesion molecule 1; HML2=macrophagelectin 2 (calcium dependent); PPP1R3C=protein phosphatase 1, regulatory(inhibitor) subunit 3C; SULT1A1=sulfotransferase family, cytosolic, 1A,phenol-preferring, member 1; MAF=v-maf musculoaponeurotic fibrosarcomaoncogene homolog (avian); BLVRB=biliverdin reductase B (flavin reductase(NADPH)); APOL1=apolipoprotein L, 1; P8=p8 protein (candidate ofmetastasis 1); PYGL=phosphorylase, glycogen; liver; FCGRT=Fc fragment ofIgG, receptor, transporter, alpha; SULT1A3=sulfotransferase family,cytosolic, 1A, phenol-preferring, member 3; AKR1C3=aldo-keto reductasefamily 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II);“sulfotransf-”=Sulfotransferase family, cytosolic, 1A,phenol-preferring, member 2; sulfotransferase family 1A,phenol-preferring, member 2 [Homo sapiens]; RRAS2=related RAS viral(r-ras) oncogene homolog 2; SERPINF1=serine (or cysteine) proteinaseinhibitor, Glade F (alpha-2 antiplasmin, pigment epithelium derivedfactor); RXRA=retinoid X receptor, alpha; C1R=complement component 1, rsubcomponent; CARHSP1=calcium regulated heat stable protein 1, 24 kDa;FST=follistatin.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique methods of inducingpancreatic hormone production has been described. Although particularembodiments have been disclosed herein in detail, this has been done byway of example for purposes of illustration only, and is not intended tobe limiting with respect to the scope of the appended claims whichfollow. In particular, it is contemplated by the inventor that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

1-34. (canceled)
 35. A method of inducing or enhancing a pancreaticislet cell phenotype or function in a population of non-pancreatic cellsprimary cells, said method comprising contacting said cell populationwith a composition comprising a nucleic acid encoding a PDX polypeptideand at least one nucleic acid encoding a beta 2, ISL-2, Nkx6.1, Ngn3.1,NKx2.2, SCG2, SGNE1, CHGN, PTPRN, AMPH, NBEA or NeuroD polypeptide in anamount sufficient to induce or enhance pancreatic islet cell phenotypeor function in said cell population.
 36. The method of claim 35, whereinsaid cell is a pluripotent stem cell.
 37. The method of claim 35,wherein said cell is a hepatic stem cell.
 38. The method of claim 35,wherein said cell population comprises a muscle cell, a spleen cell, akidney cell, a blood cell, a skin cell, a pancreas cell or a liver cell.39. The method of claim 35, wherein said cell population is provided invitro.
 40. The method of claim 35, wherein said inducing or enhancingpancreatic islet cell phenotype or function comprises inducing orenhancing at least two pancreatic islet cell characteristics selectedfrom the group consisting of an islet gene expression profile,production of a pancreatic hormone, processing of a pancreatic hormone,storage of a pancreatic hormone and secretion of a pancreatic hormone.41. The method of claim 40, wherein said pancreatic hormone is insulin,somatostatin, or glucagon.
 42. The method of claim 40, wherein saidislet cell gene expression profile comprises increased expression ofPC1/3, insulin, glucagon or somatostatin.